Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cells during a seminiferous epithelial cycle. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia have not developed into meiosis, much less through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions will facilitate molecular biology studies on pre-meiotic steps in gamete development, and provide a soma-free bioassay to identify spermatogenic factors that promote meiotic progression in vitro.