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Leydig cells, located within the interstitial compartment of the testis, are major contributors of androgen synthesis and secretion, which play an important role in testis development, normal masculinization, maintenance of spermatogenesis, and general male fertility. Accordingly, dysfunction of Leydig cells may lead to various male reproductive maladies, including primary hypogonadism, cryptorchidism, and hypospadias. A better understanding of how cell interactions and gene regulation contribute to testicular Leydig cell development and differentiation may therefore help limit the incidence of such male reproductive pathologies. Several hormones and signaling molecules have been identified as important regulators of Leydig cell differentiation and function. Recent work on the regulation of testis development, especially of Leydig cells, has focused on the Desert hedgehog and platelet- derived growth factor signaling pathways. This review outlines recent findings regarding cell interactions and gene regulation involved in the development and regulation of fetal and adult Leydig cell populations.