With the 3-1 win at home, Ajax moved up into third place in the group with three points while Man City dropped down to last place with just one point.
On Tuesday, City will look to not only avenge their loss to the Eredivisie champions but get back into the conversation about clubs that deserve to advance to the knockout round.
Group D will not be decided after the match at the Etihad Stadium but it will set the tone for the final two match days and show both clubs were they stand on the continental stage.
Here are six key battles to watch on the pitch in Manchester on Tuesday.
In the last match between the two clubs, Danish starlet Christian Eriksen was one of the top players on the pitch for Ajax.
Eriksen was a constant force in the attack for the Dutch champions and caused many problems for the Manchester City defense, and he was rewarded for his efforts with a 68th-minute goal that clinched the match.
The one man who is capable of stopping the 20-year-old in the City defense is center back and captain Vincent Kompany.
Kompany has yet again become a rock in the defense for City and will have to step up even more at home on Tuesday with his main partner in defense, Joleon Lescott, out injured.
It is likely that Matija Nastasic will start alongside Kompany at center back but with 19-year-old Nastasic still gaining experience at the top level, he will feed off of a strong performance from Kompany.
Against a talented player like Eriksen, both of the center backs for City will have to play well and shut down the Danish attacker who is the key to the Ajax attack.
Although Kenneth Vermeer and Joe Hart are going to be the two players furthest apart from each other during Tuesday's match, their impact on the match will be huge for their clubs.
Both goalkeepers have played a big part in the recent success of both clubs and Tuesday's match will see more of the same thing.
Ajax's Vermeer was the better keeper when the two clubs played at the Amsterdam Arena, and he will surely face a stronger threat from the Manchester City attack this time around.
Hart was forced to pick the ball out of his own net three times in his first meeting with Ajax, but he has not let in a goal since.
The England international will have to be in the same form he has been against Swansea and West Ham in his past two EPL matches to give City a better chance of taking down Ajax.
Ricardo van Rhijn proved two weeks ago that he is a force in the Ajax attack coming from the right-back position.
Van Rhijn was the better player on the right wing for Ajax as he outshined right-winger Tobias Sana for most of the match.
The man who will be assigned with covering Van Rhijn when he makes moves forward on the wing will be Manchester City's left-back, Gael Clichy.
Clichy has been a constant figure in the starting 11 for Roberto Mancini all season and has been the preferred left-back over Aleksandar Kolarov and Pablo Zabaleta.
Van Rhijn will have no hesitation in going forward right at Clichy if he is able to create some solid link-up play with Sana.
With the injury concerns that City are beginning to see in their defensive ranks, Clichy's good form is a pleasant sight for Mancini as he tries to figure out who plays on the right side of defense after Micah Richards' injury.
It is yet to be determined who Roberto Mancini will start up front for Manchester City but one thing is for sure, they will have to take on a strong center-back duo of Toby Alderweireld and Niklas Moisander.
In their last meeting, Mancini chose to start Edin Dzeko up front by himself with Samir Nasri, Yaya Toure and Sergio Aguero playing in the midfield positions behind the Bosnian forward.
Mancini also has the choice of playing Mario Balotelli and Carlos Tevez in those positions.
Whatever combination he chooses will have to go against one of the most underrated defensive duos in world football.
Alderweireld is finally gaining the attention he deserves after his recent performances for both club and country.
Moisander grabbed the attention of many with not only his defensive performance two weeks ago against City, but with his goal in the second half as well.
Although they may not be considered world-class by some at the moment, Alderweireld and Moisander are rapidly earning that distinction and will get closer to the world-class level with a strong match against one of the top attacks in Europe on Tuesday.
Whenever Mario Balotelli takes the pitch, he becomes a liability for Manchester City.
Balotelli is undoubtedly one of the best young attacking talents in the world, but his attitude on the pitch has sometimes prevented the 22-year-old Italian from scoring goals.
Surprisingly, the Manchester City forward has only been booked once this season, but he has also not played a full 90 minutes since September 25 against Aston Villa.
The reason for Balotelli not being on the pitch for a whole match stems from his inability to hold his emotions intact.
With the Citizens in a must-win situation, Roberto Mancini may not even let Balotelli anywhere near the pitch to avoid any bookings the Italian may earn.
But, if Balotelli is handed a start or even an appearance off the bench on Tuesday, he possesses talent that makes him a viable attacking threat especially if his club are in need of goals later in the match.
Not only did Ajax outplay Manchester City on the pitch in their last meeting but Frank de Boer out-managed Roberto Mancini as well.
De Boer's 4-1-4-1 formation caused trouble for the 4-2-3-1 that Mancini sent out at the Amsterdam Arena two weeks ago.
Mancini was aggressive with his starting lineup by starting Yaya Toure in the central attacking role in midfield instead of being one of the two defensive midfielders.
Along with starting Toure back in his regular position, Mancini must avoid going for three defenders in front of keeper Joe Hart for the whole match.
Mancini has had a tendency during the season to switch to three defenders and send a substitute on, like Aleksandar Kolarov, to go forward in attack when City are looking to win matches.
De Boer's strategy is quite simple on Tuesday, keep using the formation that works best for the club, which is the 4-1-4-1.
The center of midfield that includes Lasse Schone, Siem de Jong and Christian Poulsen is one of the strongest parts of De Boer's lineup.
Poulsen usually sets up as the player in front of the back four while Schone and De Jong play in the center of midfield.
Sticking with just one formation during a match is something De Boer will not be concerned with; for Mancini it could be the one decision that either wins the match for City or eliminates them from the Champions League.