WASp family proteins serve as conserved regulators of branched microfilament array formation via the Arp2/3 actin polymerization machinery. We have identified a specific role during spermatogenesis for the Drosophila WASp homolog (Wsp) and associated elements. Spermatogenesis within the fly testis is carried out in cysts, where a pair of somatic cyst cells encloses differentiating sperm. The final phase of the process involves the attachment of matured cysts to a specialized epithelium at the base of the testis, followed by release of individual motile spermatids into the adjoining seminal vesicle. Wsp mutant cysts contain fully mature sperm, but spermatid release does not occur, resulting in male sterility. Our data suggest that the Wsp-Arp2/3-based machinery acts in the cyst cells to influence proper microfilament organization and to enable cyst attachment to the base of the testis. Wsp activity in this context is mediated by the small GTPase Cdc42. Involvement of the cell surface protein Sticks and stones and the Wsp adapter protein D-WIP (Vrp1) is also crucial. In parallel, we demonstrate that N-WASp (Wasl), the major mammalian WASp family protein, is required in the somatic Sertoli cells of the mouse testis for sperm maturation. A requirement for WASp-based activity in somatic support cells therefore appears to be a universal feature of spermatogenesis.