Spermatogonial stem cells divide throughout life, maintaining their own population and giving rise to differentiated gametes. The unstable regulatory protein Geminin is thought to be one of the factors that determine whether stem cells continue to divide or terminally differentiate. Geminin regulates the extent of DNA replication and is thought to maintain cells in an undifferentiated state by inhibiting various transcription factors and chromatin remodeling proteins. To examine how Geminin might regulate spermatogenesis, we developed two conditional mouse models in which the Geminin gene (Gmnn) is deleted from either spermatogonia or meiotic spermatocytes. Deleting Geminin from spermatogonia causes complete sterility in male mice. Gmnn(-/-) spermatogonia disappear during the initial wave of mitotic proliferation that occurs during the first week of life. Gmnn(-/-) spermatogonia exhibit more double-stranded DNA breaks than control cells, consistent with a defect in DNA replication. They maintain expression of genes associated with the undifferentiated state and do not prematurely express genes characteristic of more differentiated spermatogonia. In contrast, deleting Geminin from spermatocytes does not disrupt meiosis or the differentiation of spermatids into mature sperm. In females, Geminin is not required for meiosis, oocyte differentiation, or fertility after the embryonic period of mitotic proliferation has ceased. We conclude that Geminin is absolutely required for mitotic proliferation of spermatogonia but does not regulate their differentiation. Our results suggest that Geminin maintains replication fidelity during the mitotic phase of spermatogenesis, ensuring the precise duplication of genetic information for transmission to the next generation.