Spermatogonial stem cell (SSCs) proliferate and differentiate to sperm in seminiferous tubules. SSCs are under well-organized control of germ cell niche composed of Sertoli cells, Leydig cells, peritubular myoid cells (PMCs) and vasculature. Thus, Sertoli cell dysfunction causes spermatogenic failure. Recently we discovered benzalkonium Chloride (BC) ablates Sertoli cells selectively. We tried manipulating mouse germ cell niche by seminiferous transplantation into BC treated testis. FACS sorted donor Sertoli cells colonize and support recipient spermatogenesis. When we transplanted whole testicular cells into Sertoli-depleted testis, donor SSCs, Sertoli, Leydig and PMCs colonize and reconstruct donor testis in recipient testis. BC has been used clinically for decades and ablates mouse and canine Sertoli cells, suggesting that this novel method of drug-induced Sertoli cell elimination followed by replacement may be useful for male infertility patients with Sertoli cell disorder. Using this novel technique, we can reorganize mouse germ cell niche. This might achieve human testis reconstruction in mouse testis and can be a strategy of fertility preservation for young cancer patients.