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It is heavily implied at in the game that Harley Quinn is or was pregnant. This is suggested by examining Harley's costume on a dummy and panning down to the floor. At the dummy's feet is a box reading "Fruitful Endeavors pregnancy test". Lying beside it is a positive test. Also during the end credits, Harley can be heard singing an alternate version of "Hush, Little Baby."
During the Catwoman DLC mission to get her loot back from Two-Face, Two-Face says, "Whoever kills the Cat gets to be my number one... guy" in a manner very similar way to how Jack Nicholson's Joker says to Bob (the goon) "And remember... you... are my number one... guy" in the Tim Burton's Batman (1989) movie.
There are several references to Lost (2004), the ABC television show. One when flying around Arkham City, an inmate asks another, "So, did they ever explain what the island was?" And later, more dialogue: "...why did it end in a church?" Writer Paul Dini was part of the "Lost" writing staff for the first two seasons before leaving the series.
An Easter egg/reference relating to The Scarecrow can be found on a barge in the docks area. When on the barge, use the sequencer gadget to solve the code "City of Terror". A panel opens. Once inside, you are in a room filled with neon bug zappers. At the end of the hall, there is an unconscious inmate sitting in a chair. Upon zooming in on him, he is startled awake and gives a loud shriek. He goes back to sleep. It is assumed that he is a victim of The Scarecrow's experiments.
Rocksteady decided not to use the Batmobile in this game due to Arkham City's rugged terrain and narrow environments.
This is the first known project where Tara Strong has voiced the role of Harley Quinn, which has predominantly been voiced by creator Paul Dini's friend Arleen Sorkin. Strong is better known in the Batman animated universe in the role of Barbara Gordon, aka Batgirl and in this game aka Oracle.