The preparation of spermatogonial stem cell (SSC) transplant recipients laid the technical foundation for SSC transplant technology and the understanding of spermatogenesis mechanisms. Busulfan is commonly used to prepare recipients for mouse SSC transplantation; however, its safety and efficiency have been questioned. This review summarizes the relationship between SSCs and Sertoli cells (SCs), and the mechanism of busulfan toxicity against sperm cells. We concluded that the proliferation, differentiation, and apoptosis of SSCs are regulated by SCs. The endogenous spermatogenic cells are depleted by busulfan treatment via alkylation of DNA, destruction of vimentin filament distribution, disruption of SSC differentiation, promotion of SSC dormancy, and generation of oxidative stress. However, the mechanisms require further exploration. The recent establishment of a model in vitro culture system has provided a good technical foundation to further explore these mechanisms, which will help us to find more efficient methods of recipient preparation and optimal transplantation times.