The biological activities of spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and thus sustained male fertility. Therefore, understanding the mechanisms governing their ability to both self-renew and differentiate is essential. Moreover, because SSCs are the only adult stem cell to contribute genetic information to the next generation, they are an excellent target for genetic modification. In this chapter, we discuss two important approaches to investigate SSCs and their cognate niche microenvironment in the mouse, the SSC transplantation assay and the long-term serum-free SSC culture method. These techniques can be used to enhance our understanding of SSC biology as well as to produce genetically modified animals.