Frank Lillie and Alfred Jost, the two pillars in the field of reproductive sciences, laid the foundations of modern knowledge on sexual differentiation. Lillie’s description of Freemartin, a masculinized female calf born as a twin with a male calf, pioneered the hypothesis that shaped the concept of hormonal influence on sex determination: The masculinized reproductive tracts and gonads of the female twin is caused by diffusible factors or hormones that come from the male twin through the placenta. Alfred Jost further advanced the filed by establishing the paradigm of sexual differentiation, based on his discovery that testis-derived androgen in the male embryo is responsible for the maintenance of Wolffian duct, the precursor of male reproductive tract. In the female embryos, on the other hand, their reproductive systems arise by default as a result of a lack of androgens. In the past few years, my laboratory revisited Lillie’s freemartin model and Jost’ paradigm using modern mouse genetic approaches. In this presentation, I will discuss our efforts on identifying the freemartin factors and redefining the concept of the Jost paradigm.