Maintenance of fertility in adult males is dependent on a pool of germline stem/progenitor cells (spermatogonial progenitor cells; SPCs) within the testis that continually generate differentiating germ cells for the production of spermatozoa. SPC self-renewal in the mouse is critically dependent upon expression of Promyelocytic leukemia zinc finger (Plzf), a transcription factor originally identified from its involvement in human leukemia. However, the distinct downstream targets of Plzf that are essential for regulation of SPC maintenance remain poorly defined. Spalt-like 4 (Sall4) is a transcription factor essential for early development and embryonic stem cell function. In adults, Sall4 expression is largely restricted to the gonads suggestive of a specific role within germ cells. Importantly, we have found Sall4 to be highly expressed in SPCs and to physically interact with Plzf. Further, Sall4 and Plzf can mutually oppose one another’s function and localization to cognate chromatin domains. Through conditional deletion of Sall4 within the germline we demonstrate key roles for this factor in embryonic germ cell maintenance and subsequent differentiation of SPCs; distinct roles to that of Plzf. In addition, we show that Sall4 and Plzf antagonistically co-regulate Kit and Sall1, genes associated with SPC differentiation and maintenance respectively. Based on our data we propose that a balance between directly opposing Sall4 and Plzf activities regulates the switch from SPC self-renewal to differentiation and suggest a new model for formation of stable adult stem cell pools.