Research on in vitro spermatogenesis has a long history and remained to be an unaccomplished task until very recently. In 2010, we succeeded in producing murine sperm from primitive spermatogonia using an organ culture method. The fertility of the sperm or haploid spermatids was demonstrated by microinsemination. This organ culture technique uses the classical air-liquid interphase method and is based on conditions extensively examined by Steinbergers in 1960s. Among adaptations in the new culture system, application of serum-free media was the most important. The system is very simple and easy to follow.