Yep, it’s when they go into that store. The owner assumes they’re together which I completely understand:
SHOPKEEPER: Help you, boys?
DEAN: Hope so. Uh, we were playing Jenga over at the Walshes’ the other night, and, uh… well, he hasn’t shut up since about this Christmas wreath, and (to SAM) I don’t know, you tell him.
SAM: (Giving DEAN a look) Sure. (turns back to the shopkeeper) It was yummy.
SHOPKEEPER: I sell a lot of wreaths, guys.
SAM: Right, right, but – but you see, this one would have been really special. It had, uh, it had, uh, green leaves, um, white buds on it. It might have been made of, uh… meadowsweet?
SHOPKEEPER: Well, aren’t you a fussy one?
SAM looks taken aback, embarrassed.
DEAN: (Smiles fondly) He is… (He laughs and SAM looks at him)
Or were you asking why the shopkeeper guy thought they were gay? Aside from the way Dean starts talking about their game night with another couple, I’d say it’s probably this business right here:


If I was playing a fun game called Are They Brothers or a Are They a Gay Couple?, I know what my guess would be after seeing these looks.