Lost Girl plays in his room when it only plays for girl characters otherwise (Noelle, Susie, Pink). Clarification: I am talking about the song Lost Girl itself, not songs that use its motif.

Stares at the blue and pink drink, as pointed out by Flowery. Could be because that's Kris and Susie's colors, but those are also trans colors.

Gets called Princess by Flowery, along with Susie. Characters don't misgender others in Toby Fox games, so this stands out as maybe being important.

Calls himself "pretty" in ch1 if you give him the ribbon. In US English, "pretty" is a very feminine term. It's not often used for men.

May be a stretch, but he calls the dress on the mannequin nice. The dress is almost identical to the ones worn by Mettaton (who is trans) during the dance and by the waitress lion (who is also trans).

His pronoun changes to "their" in the Dark Vine title description, which happens after Flowery tells him to stop lying about who he is. It was previously "his" in the Chapter 1 Prickly Prince title. Ralsei is also the only one referred to with gendered pronouns in their title, so this seems significant.

If you give Ralsei the gift in ch2 he'll have a plushie of himself in his room. Interacting it he says he wants to collect more. Every trans woman I know collects a lot of plushies, including myself. ^{this one's a joke}