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Aggressive chemotherapy may lead to permanent male infertility. Prepubertal males do not generate sperm, but their testes do contain spermatogonial cells (SPGCs) that could be used for fertility preservation. In the present study, we examined the effect of busulfan (BU) on the SPGCs of immature mice, and the possible induction of the survivor SPGCs to develop spermatogenesis in 3D in-vitro culture. Immature mice were injected with BU, and after 0.5&ndash, 12 weeks, their testes were weighed and evaluated histologically compared to the control mice. The spermatogonial cells [Sal-like protein 4 (SALL4) and VASA (a member of the DEAD box protein family) in the testicular tissue were counted/seminiferous tubule (ST). The cells from the STs were enzymatically isolated and cultured in vitro. Our results showed a significant decrease in the testicular weight of the BU-treated mice compared to the control. This was in parallel to a significant increase in the number of severely damaged STs, and a decrease in the number of SALL4 and VASA/STs compared to the control. The cultures of the isolated cells from the STs of the BU-treated mice showed a development of colonies and meiotic and post-meiotic cells after four weeks of culture. The addition of homogenates from adult GFP mice to those cultures induced the development of sperm-like cells after four weeks of culture. This is the first study demonstrating the presence of biologically active spermatogonial cells in the testicular tissue of BU-treated immature mice, and their capacity to develop sperm-like cells in vitro.