Spermatogonial stem cells sustain spermatogenesis within a Sertoli cell-organized niche, yet xenogeneic spermatogenic differentiation after transplantation into host mouse seminiferous tubules has been reported in only a limited number of donor species. Here, we tested whether host niche engineering could support xenogeneic spermatogenic differentiation. Immunodeficient mouse testes were preconditioned by busulfan-mediated germ cell depletion followed by intratubular benzalkonium chloride treatment to reduce Sertoli cells, and cryopreserved rat testicular cells were transplanted into the modified tubules. Donor-derived germ cells and Sertoli cells colonized the basement membrane, reconstructed seminiferous tubule-like structures, and supported advanced spermatogenic differentiation, including elongating spermatids. These findings show that frozen-thawed donor testicular cells can organize seminiferous tubule-like structures across species boundaries and provide a framework for investigating engineered testicular niches.