Sertoli cells are supporting cells of the testicular seminiferous tubules, which provide a nurturing environment for spermatogenesis. Adult Sertoli cells are polarized so that they can simultaneously support earlier-stage spermatogenic cells (e.g., spermatogonia) basally and later-stage cells (e.g., spermatids) apically. To test the consequences of disrupting cell polarity in Sertoli cells, we perform a Sertoli-specific conditional deletion of Rac1, which encodes a Rho GTPase required for apicobasal cell polarity. Rac1 conditional knockout adults exhibit spermatogenic arrest at the round spermatid stage, with severe disruption of Sertoli cell polarity, and show increased germline and Sertoli cell apoptosis. Thus, Sertoli Rac1 function is critical for the progression of spermatogenesis but, surprisingly, is dispensable for fetal testicular development, adult maintenance of undifferentiated spermatogonia, and meiotic entry. Our data indicate that Sertoli Rac1 function is required only for certain aspects of spermatogenesis and reveal that there are distinct requirements for cell polarity during cellular differentiation.