Spermatogenesis refers to a complex differentiation process by which diploid spermatogonial stem cells develop into mature haploid spermatozoa. Sertoli cells (SCs), the only somatic constituents of the seminiferous epithelium, are in direct physical association with all types of germ cells (GC) and are mainly committed to sustain spermatogenesis. Importantly, individual SC can only support a finite number of germ cells. Therefore, the final testis size, the number of germ cells in the adult testis, and sperm output are directly linked to the total number of SCs. Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating Sertoli cell survival, proliferation and maturation remain incomplete. To investigate whether the insulin-like growth factor (IGF) system is required for germ cells (GCs) and Sertoli cells (SCs) development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r) or both receptors specifically in the GC lineage or in SCs. While ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SC during the late fetal and early neonatal testicular period. In addition in vivo and analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size and daily sperm output. It also indicates that the insulin/IGF signaling pathway is required for FSH-mediated Sertoli cell proliferation.