Continual spermatogenesis is the cornerstone of male fertility and relies on the actions of an undifferentiated spermatogonial population comprised of stem cells and progenitors. A foundational spermatogonial stem cell (SSC) pool is established during postnatal development that serves as a self-renewing reservoir from which progenitor spermatogonia arise that transiently amplify in number before committing to terminal differentiation. At present, the underlying molecular mechanisms governing these actions are undefined. Using conditional mutant mouse models, we investigated whether function of the undifferentiated spermatogonial population during postnatal life is influenced by the tumor suppressor protein RB1. Spermatogenesis initiates in mice with conditional inactivation of Rb1 in prospermatogonial precursors, but the germline is progressively lost upon aging due to impaired renewal of the undifferentiated spermatogonial population. In contrast, continual spermatogenesis is sustained following Rb1 inactivation in progenitor spermatogonia, but some cells transform into a carcinoma in situ-like state. Furthermore, knockdown of Rb1 abundance within primary cultures of wild-type undifferentiated spermatogonia impairs maintenance of the SSC pool, and some cells are invasive of the basement membrane after transplant into recipient testes, indicating acquisition of tumorigenic properties. Collectively, these findings indicate that RB1 plays an essential role in establishment of a self-renewing SSC pool and commitment to the spermatogenic lineage within progenitor spermatogonia.