Hmmm. Hard to say. Looking back I can see that in many ways my childhood best friend treated me pretty much like her boyfriend. And before the dawn of puberty, and experiencing my first real crush on a boy, I was pretty comfortable in that role. Even in our early teens she tried to get me to make out with her, although to my knowledge she is, to this day, completely straight.
My grandmother's 2nd husband gave me a tux when I was 4 or 5. I'm not sure if he sensed something, or if it just didn't fit the intended recipient, and I was the right size. My grandmother died shortly after that, and I never saw him again, so I have no other interactions to judge from.
And there was the boy in my 5th grade class who said he liked me because I "Don't care if someone is a boy or a girl." I got the impression at the time that he meant I acted as much like a boy as like a girl.
I remember when I was about 12 and finally won the battle to choose all of my own clothes, I was occasionally mistaken for a boy, despite having hair down to my waist. In fact, I remember distinctly one day in the video store a little kid asking me if I was a boy or a girl. I remember feeling very uncomfortable with the question because of the battle between the answer that was technically true and the answer that felt true.
I now also realize that one of my co-workers when I was in my early 20's almost certainly knew. I remember she dropped several casual mentions of trans-guys she knew. At one point she loaned me a book, saying that the protagonist's sister made her think of me, and she thought I'd like it. The sister was a girl in her early teens who posed as a boy in order to be a drummer in a Civil War reenactment group. (At the time I was a drummer at a Renaissance Faire, so I convinced myself that that was all my co-worker was referring to.)
As an adult I was mostly mistaken as Lesbian, to the extent that some people reacted with disbelief when they found out I liked men.

Come to think of it, I did have a couple friends in high school ask me, in a round about way, if I was gay.
But I don't have any instances where anyone came out and said, "You're really a boy, aren't you?"