Members of the Nuclear Factor I family (NFI) are key regulators of stem cell biology during development, with well documented roles for NFIA, NFIB and NFIX in a variety of developing tissues, including brain, muscle and lung. Given the central role these factors play in mediating stem cell biology in various systems, we posited that they might also be pivotal for spermatogonial stem cells during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, we revealed that three NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonial stem cells, as we anticipated, but in pre-meiotic spermatocytes. These data suggested a potential role for NFIX in spermatogenesis so we investigated mice with constitutive deletion of Nfix (Nfix-/-). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-/- mice (Nfix-/- mice do not survive past P22) revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. We found that many developing spermatocytes exhibited multinucleation, cytokinetic defects, as well as a significant increase in the number of apoptotic cells in the Nfix-/- testes, compared to controls. As a result of these defects, spermatogenesis arrests at early diplotene and very few round spermatids were observed. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.