Freud (1905/1953) anchored his theories of unconscious psychological functioning in observations and inferences about childhood sexuality. These ideas remain influential among psychoanalysts today. Much progress subsequently occurred in extra-psychoanalytic research in human sexuality. This included the discovery in 1959 of an entirely new area of psychology: the sexual differentiation of behavior (Phoenix, Goy, Gerall, & Young, 1959; Wallen, 2009). The observations that led to this new field originally concerned the effects of androgen administered prenatally to non-human animals. This early research was compatible with later studies of humans as well. Prenatal androgen influences both erotic and non-erotic behavior, including childhood rough-and-tumble play (RTP). We have previously emphasized the need to integrate this psychoneuroendocrine knowledge with psychoanalytic theory and practice (Friedman, 1988; Friedman & Downey, 2002, 2008a, 2008b). In this article, we discuss additional aspects of the relationship between sexually differentiated childhood play, particularly RTP, and gender differences in psychosexual development and functioning. These topics and other aspects of the sexual differentiation of behavior have been under-emphasized in psychoanalytic thought.