It is heavily implied at one point 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.
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.
When you go to Two Face's courthouse and you enter the back way; you can go down to the cell next to the Calendar Man's cell. When you're in that cell turn on detective mode, look up (make sure you are under them), and punch. When you do that they somehow notice you and run through the door and come to fight you.
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.
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.
During the Catwoman 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 to Jack Nicholson's Joker. Joker says to Bob in the Burton Movie "And remember... you... are my number one... guy."
Peter MacNicol voices The Madhatter - a villain obsessed with collecting Batman's cowl. MacNicol also voices X The Eliminator - a villain similarly obsessed with collecting part of the hero's outfit (his crest) - In Harvey Birdman Attorney At Law.
The trivia items below may give away important plot points.
After the release of this game, Mark Hamill announced he was retiring from voicing the Joker. However, he said that if an adaption of the graphic novel "Batman: The Killing Joke" was ever released, he would return to the role. Batman: Arkham Knight featured a flashback to this event, and Mark Hamill returned as The Joker for it.
In the end of the third Catwoman DLC mission, the player is presented with a choice: Leave Arkham City with her loot, or leave it behind in the vault, but save Batman who has become trapped in the Steel Mill. On the floor, a green arrow points towards where the "better" option of the two is (save Batman), and likewise for the red arrow pointing towards the "lesser" choice (leave Arkham City).
Before you realize there is a fake joker, you can hear Harley Quinn say something along the lines of "Oh Mistah J you look perfect! Oh wait it isn't you is it?" And joker says "Be quiet Harley! This is a foreshadowing hint that Clayface impersonates Joker later on.
Find either a balcony or a bill board and spam freeze grenades. You will start to float in the air. If you go high enough the TYGER guards guarding the wall on the edge of arkham city will not spot you and you are free to roam gotham city.
During the joker boss fight that takes place a few minutes before Protocol 10, if you switch to detective mode while fighting him, you can see that he doesn't have a skeleton while the thugs and lieutenant do. This is one of the many hints given throughout the main storyline that Joker is just being imitated by Basil Karlo, AKA Clayface.