Spermatogonia represent a new route to transgenesis in mice and potentially in some commercially important domesticated animals. In addition, these cells are also a potential target for viral integration in patients receiving somatic cell gene therapy. But the factors influencing retroviral transduction into spermatogonia are not well understood. Because retroviral transduction is affected in part by the proliferative status of the host cell, we developed an improved cell culture system in which spermatogonia survive and proliferate for several days. We used this system to test the ability of a variety of murine and avian retroviruses to infect spermatogonia. We investigated the factors influencing retroviral transduction of spermatogonia, including the proliferative status of the infected cell, the type of viral envelope, the type of retroviral long terminal repeat, and the method of viral delivery. Here we show that many of the widely used retroviral vector systems can be used to successfully transduce spermatogonia at high efficiency. Moreover, we show that retroviral delivery of MDM2, the major downregulator of p53, promotes spermatogonial survival in culture, suggesting that p53 plays a role in regulating spermatogonial apoptosis induced by growth factor deprivation. These results further demonstrate the usefulness of this novel system of targeting substances of interest to the testis. These data have important implications for improving animal transgenesis and for understanding the risks associated with somatic cell gene therapy.