Connexin 43 (CX43, also known as GJA1) is the predominant testicular gap-junction protein. In the seminiferous epithelium, gap junctions occur between adjacent Sertoli cells and between Sertoli and germ cells. The specific role of CX43 in Sertoli cell has been difficult to study because of the neonatal lethality of the Cx43 global knockout. Recently two laboratories have independently developed a Sertoli cell conditional Cx43 knockout using Cre-loxP methodology. These have allowed the role of CX43 in Sertoli cells to be determined while circumventing lethal abnormalities in organs such as the heart seen in Cx43 global knockouts. In this review the focus is on the insights into the role of CX43 in Sertoli cells derived from these new model systems. Results indicate that CX43 is essential for cessation of proliferation and normal maturation in Sertoli cells. In addition, CX43 in Sertoli cells is required for spermatogenesis, but not early germ cell proliferation. These model systems show that CX43 is important in Sertoli cell development, and will be useful in further studies of its role in Sertoli cell and testis biology.