Spermatogonia, which produce sperm throughout the male lifetime, are regulated inside a niche composed of Sertoli cells, and other testis cell types. Defects in Sertoli cells often lead to infertility, but replacement of defective cells has been limited by the inability to deplete the existing population. Here, we use an FDA-approved non-toxic drug, benzalkonium chloride (BC), to deplete testis cell types in vivo. Four days after BC administration, Sertoli cells are preferentially depleted, and can be replaced to promote spermatogenesis from surviving (host) spermatogonia. Seven days after BC treatment, multiple cell types can be engrafted from fresh or cryopreserved testicular cells, leading to complete spermatogenesis from donor cells. These methods will be valuable for investigation of niche-supporting cell interactions, have the potential to lead to a therapy for idiopathic male infertility in the clinic, and could open the door to production of sperm from other species in the mouse.