Ionae's subplot, where she gives heartfelt advice to a group of Emo Teens to help them get over their issues, then saying "I Choose to Stay" when given a choice of going back to the Counterplanes, because she grew a conscience.
The game's main theme that is frequently quoted in other tracks, A Lost Dream. It's a pity it only appears in the opening scene, where most players probably won't wait to hear all of it.
Demonic Spiders: The combat is kept fresh by giving almost every enemy or set of enemies a new or unusual ability or gimmick. This leads to many enemies who will induce hair-pulling for the unprepared. There are also some who simply have such strong armor or self-healing abilities that hurting them significantly takes a huge effort.
Fridge Brilliance: It initially seems very confusing why the scientific research station in Chapter 6 is in such an obscure location and why no major breakthroughs have been made by all the (admittedly kooky) scientists there. Then you realize this is how the Rakari isolate and supervise scientific minds so they can mind wipe and suppress any discoveries to preserve their Medieval Stasis.
Tear Jerker: If you have Charlotte in your party, at the beginning of chapter 7, when the Keepers ask her about Isabelle, she starts talking about her life story in a way that is nothing short of heartbreaking, especially if you imagine her saying it in a Creepy Monotone because of the herbal intoxicant the Keepers gave her.
One that you'll see regardless of which characters you chose: Once you finally get to the World Eye, you'll find a critically damaged Clay 13 holding Shara's limp body. He says "[Batiste] broke her and now I can't make her move." When your party members say that she's dead, he says "I don't understand. You can make her smile again! The others would laugh at me, but she was always...always...so...kind..." Before dying himself.
Some of the Hannibal LecturesIck Thelloth delivers in chapter 8 can be pretty tear jerking as well, especially Charlotte's. Also, possibly an in-universe example, as your characters will go into a "depressed" state afterwards.
That One Boss: Ick Thelloth, who reflects a portion of all damage he receives onto one of your party members (which cannot be dodged or cheated in any way), and has extremely powerful attacks, only some of which can be defended against. Don't try to beat him on hard mode unless you've got an hour or so blocked out for the task.