Early 1990s research conducted by Peter Koopman, John Gubbay, Nigel Vivian, Peter Goodfellow, and Robin Lovell-Badge, showed that chromosomally female (XX) mice embryos can develop as male with the addition of a genetic fragment from the Y chromosome of male mice. The genetic fragment contained a segment of the mouse Sry gene, which is analogous to the human SRY gene. The researchers sought to identify Sry gene as the gene that produced the testis determining factor protein (Tdf protein in mice or TDF protein in humans), which initiates the formation of testis. Koopman's team published their results in 1991 in Male Development of Chromosomally Female Mice Transgenic for Sry gene. Their results showed that Sry gene partly determines the sex of an embryo and is the only gene on the Y chromosome necessary for initiation of male development in mice.

Frank Rattray Lillie's research on freemartins from 1914 to 1920 in the US led to the theory that hormones partly caused for sex differentiation in mammals. Although sometimes applied to sheep, goats, and pigs, the term freemartin most often refers to a sterile cow that has external female genitalia and internal male gonads and was born with a normal male twin. Lillie theorized that a freemartin is a genetic female whose process of sexual development from an undifferentiated zygote was suppressed or antagonized by her twin's release of male hormones via their shared blood circulation in utero. Despite publications of similar findings by physician Julius Tandler in Vienna, Austria, in 1910 and physician Karl Keller in Wiesensteig, Germany in 1916 prior to Lillie's research, Lillie often receives credit for the hormonal theory of sex differentiation in the freemartin. Lillie's study of freemartins, and the subsequent research by graduate students in Lillie's laboratory at the University of Chicago in Chicago, Illinois, prompted many embryologists to research sex differentiation and hermaphroditism in mammals.

In 1947, Carl Richard Moore, a researcher at the University of Chicago, in Chicago, Illinois, wrote Embryonic Sex Differentiation and Sex Hormones, which was published in the same year as a first-edition monograph. In the book, Moore argues that regulation of sex differentiation in mammals is not controlled by sex hormones secreted by embryonic sex organs (gonads), but is controlled by non-hormonal genetic factors. In support of his hypothesis, Moore describes the current literature on sex differentiation, and he reviews experiments on vertebrates and invertebrates and his own work with opossum (Didelphis virginiana) young.