Sox3, a member of the high mobility group (HMG) family of transcription factors, is expressed in neural progenitor cells and in the gonads. Targeted deletion of Sox3 in mice causes abnormal development of the diencephalon and Rathke's pouch, the progenitor of the anterior pituitary gland. Male and female mice are also infertile and exhibit a primary defect in gametogenesis. In this study, we examined the expression and function of Sox3 in C57BL/6 mice to better understand its role in spermatogenesis. Testis development was normal during embryogenesis. However, spermatogenesis failed to progress during the postnatal period, with germ cell loss beginning at postnatal day 10 (P10). By P14, Sox3 null mice were nearly agametic, retaining only Sertoli cells and undifferentiated spermatogonia. Pituitary gonadotropin and testosterone levels were normal, suggesting a defect in Sertoli cell and/or germ cell function. Immunostaining revealed that Sox3 was expressed in a subpopulation of germ cells localized at the base of the seminiferous tubules. Sox3 expression was restricted to proliferating germ cells and colocalized with neurogenin 3 (Ngn3), a helix–loop–helix transcription factor implicated in spermatogonial differentiation. The absence of Sox3 decreased Ngn3 and increased expression of Oct4, a marker of undifferentiated spermatogonia. We conclude that Sox3 is expressed in A_s, A_pr and A_al spermatogonia and is required for spermatogenesis through a pathway that involves Ngn3.