Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I⁻ Thy-1 α6-integrin αv-integrin^−/dim throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.