The development of experimental models treated by chemotherapy is needed for elucidating the side effects of cancer treatments administered prior to puberty on male gonad function and the feasibility of restoring fertility from exposed testicular tissues. This study investigated for the first time the effects of cytarabine and daunorubicin administered before meiotic initiation on the first wave of mouse spermatogenesis under both in vivo or in vitro conditions. Prepubertal exposure to cytarabine did not exhibit immediate detrimental effects on testicular tissues, whereas daunorubicin administration resulted in a decreased spermatogonia-to-Sertoli cell ratio and diminished intratubular cell proliferation within three days post-treatment. While the completion of in vivo spermatogenesis was not hindered by chemotherapy exposure, a significant increase in the proportion of spermatozoa with fragmented DNA was observed in mice more than one month after treatment. In vitro spermatogenesis was also accomplished using prepubertal testicular tissues exposed to chemotherapy, indicating that neither cytarabine nor daunorubicin impeded the differentiation potential of spermatogonia into spermatozoa. However, in vitro conditions revealed an arrest in meiotic progression in a substantial proportion of seminiferous tubules and an elevated incidence of DNA double-strand breaks in intratubular cells compared to in vivo controls, irrespective of the treatment administered.