Spermatogonial stem cells (SSCs) produce sperm throughout the post-pubertal life of a male. Transgenic loss- and gain-of-function mouse models have shown that their self-renewal and differentiation are controlled in vivo by glial-cell-line-derived neurotrophic factor (GDNF) in a dose-dependent manner. After this in vivo observation, the culture conditions for mouse SSCs were rapidly developed. GDNF together with other growth factors, hormones, and vitamins maintain proliferation and self-renewal of SSCs for years in vitro. Both serum-supplemented and serum-free culture methods have been described. The cells are cultivated either on feeder layer or laminin-coated dishes. First reports from random and targeted mutagenesis of SSCs have been published. Some cells in the spermatogonial stem cell culture transform to embryonic stem cell-like cells and form teratomas in nude mice. In general, the spermatogonial stem cells maintain their germline identity in long-term culture. The mechanism for transformation to embryonic stem cell-like cells is not known, but the data suggest that germline and embryonic stem cells are closely related. We describe in detail the culture system of SSCs developed by Dr. Takashi Shinohara in 2003.