In adult males, spermatogonial stem cells function to replenish developing gametes that are continuously released from the testes as mature spermatozoa. Because of their potential importance to research, medicine, industry, and conservation, numerous attempts have been made in the past to cultivate sperma-togonial stem cells in vitro. However, only recently have culture methods been established that effectively promote the proliferation of mammalian spermatogonial stem cells in vitro. We describe a simple and reproducible protocol for the derivation and maintenance of mouse spermatogonial stem cell lines that proliferate for long periods of time in culture.