Germ cells occupy a unique place in development through their requirement to maintain underlying totipotency while producing highly differentiated gametes. This is reflected in the expression of key regulators of pluripotency in the fetal germline and the ability of these cells to form pluripotent stem cells and germ cell tumors. Culture of whole fetal testes, including germ cells, provides a key model for studying gonad and germline development, but it is critical that such models mimic physiological development as closely as possible. We aimed to determine the effects of differing culture conditions, including serum-free and serum-containing conditions, on fetal germ cell and testis development. We tested a commonly used model that employs knockout serum replacement (KSR) to provide more defined culture conditions than media containing fetal bovine serum (FBS). In FBS conditions, cell cycle parameters in germ and Sertoli cells closely resembled normal development. In contrast, KSR significantly inhibited male germ cell entry into mitotic arrest, a key milestone in male germline development. Moreover, KSR disrupted molecular control of cell cycle and inhibited the transcription of a range of male germ cell differentiation markers. In the somatic compartment, KSR stimulated proliferation and inhibited differentiation in Sertoli cells. These data demonstrate that KSR substantially alters germ and somatic cell differentiation in fetal testis culture and should not be used to replicate normal gonadal development. In contrast, basal media with or without serum support germ and supporting cell differentiation and cell cycle dynamics that are in line with in vivo characteristics.