In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present in both Sertoli cells (SC) and germ cells (GC). In order to determine the relative contributions of each source of ATRA, we have generated mice lacking all RALDH activities in the seminiferous epithelium (SE). We show that both the SC- and GC-derived sources of ATRA cooperate to initiate and propagate spermatogenetic waves at puberty. In adults, they exert redundant functions and, against all expectations, the GC-derived source does not perform any specific role despite contributing to two-third of the total amount of ATRA present in the testis. The production from SC is sufficient to maintain the periodic expression of genes in SC, as well and the cycle and wave of the SE which account for the steady production of spermatozoa. The production from SC is also specifically required for spermiation. Importantly, our study shows that spermatogonia differentiation depends upon the ATRA synthesized by RALDH inside the SE, while initiation of meiosis and expression of STRA8 by spermatocytes can occur without ATRA.