In the adult mammalian testis, spermatogenic differentiation starts from a minute population of spermatogonial stem cells (SSCs). SSCs are generated after birth from the fetal gonocytes, themselves derived from the primordial germ cells (PGCs), which are specified during the first days after implantation. Transcriptome profiling of purified preparations evidenced the preferential accumulation in SSCs of transcripts of PU.1 (Sfpi1), a regulatory gene previously identified in hematopoietic progenitors. In situ immunolabeling and RNA determination showed a complex pattern of expression in the adult testis, first in SSCs and early spermatogonia followed by de novo expression in pachytene spermatocytes. Spermatogenesis in a null mutant (PU.1(G/G)) was arrested at the prenatal stage, with reduced numbers of gonocytes owing to a defect in proliferation already noticeable at E12.5. Transcripts of several germinal markers, including vasa (Mvh, Ddx4), Oct4 (Pou5f1), Dazl and Taf4b, were detected, whereas stella (PGC7, Dppa3) was not. Germ cells of PU.1(G/G) newborn testes grafted in nude mice did not initiate the postnatal replicative stage, whereas grafts of their wild-type littermates underwent complete spermatogenesis. During embryonic development, PU.1 transcription was initiated as early as the blastocyst stage, with a generalized expression at E6.5 in the embryonic ectoderm. PU.1 therefore appears to play a determinant role in at least two distinct lineages and, given its wide range of expression, possibly in other stem cells.