Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.