Accumulating evidence suggests that chemotherapy can cause long‐term detrimental effects and alter the biology of the recipient environment. Therefore, a subsequent report claimed that the transplantation of female germline stem cells (FGSCs) into the ovaries of recipient mice that were pretreated with a high dose of busulfan and cyclophosphamide (B/C) resulted in the successful production of offspring. Therefore, this study was conducted to further clarify the impact of female germ cell transplantation on female ovaries after B/C treatment. RT‐PCR analysis showed that the period of germ cell depletion coincide with decreased Figla, Lhx8, Nobox, Kit, and Sox3 gene expression in the B/Ctreated ovary. However, depletion of female germ cells is mediated by a Fas/FasL‐, Kit/ Kitl‐, TNF‐, p53‐ and autophagy‐ independent pathway. Also, histological analysis is similar to that of Nobox null‐derived ovaries, indicating that follicle death after B/C treatment might be caused by down‐regulating of Nobox pathway. When female mice during 15 weeks after B/C treatment were checked for reproductive activity, B/C treated mice did not produce their pups. In addition, when 3×106 GFP positive primordial follicles were injected into B/C treated female mouse ovaries, donor follicle were not able to colonize into the ovaries of recipients. In conclusion, these data from a preclinical mouse model strongly suggested that female ovary until 15 weeks after B/C treatment could not support environment for maturing of exogenous FGSCs.