Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture multiple stages of germ cells during spermatogenesis. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, is vital for spermatogenesis. However, the mechanisms and developmental timing that underlie Sertoli cell polarity are poorly understood. We investigate this aspect of testicular function by conditionally deleting Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Sertoli Cdc42 deletion leads to increased apoptosis and disrupted polarity of juvenile and adult testes but does not affect fetal and postnatal testicular development. The onset of the first wave of spermatogenesis occurs normally, but it fails to progress past round spermatid stages, and by young adulthood, conditional knockout males exhibit a complete loss of spermatogenic cells. These findings demonstrate that Cdc42 is essential for Sertoli cell polarity and for maintaining steady-state sperm production.