Testicular spermatogonial stem cells (SSCs) are a heterogeneous population of stem cells, and definitive marker for the most primitive subset that undergoes asymmetric cell division remains to be identified. A novel subpopulation of pluripotent, very small embryonic-like stem cells (VSELs) has been reported in both human and mouse testes. Follicle-stimulating hormone (FSH) receptors (FSHRs) are expressed on Sertoli cells in testis and on granulosa cells in ovary, but recently FSHRs are reported on VSELs in ovaries, bone marrow, and cord blood. The present study was aimed to investigate whether FSHRs are also expressed on testicular stem cells (VSELs and SSCs) and their possible modulation by FSH using intact and chemoablated (25 mg/kg busulfan) mice. Chemoablated testis was a better model to study stem cell biology since quiescent stem cells survive along with the Sertoli cells in the tubules. Proliferating cell nuclear antigen-positive, small-sized cells presumed to be VSELs were clearly visualized, and flow cytometry analysis revealed an increase in LIN-/CD45(-)/SCA-1(+) VSELs from 0.045 +/- 0.008% to 0.1 +/- 0.03% of total cells in chemoablated testis after FSH treatment. Very small embryonic-like stem cells expressing nuclear octamer-binding transcription factor 4 (OCT-4) and SSCs with cytoplasmic OCT-4 were detected. Very small embryonic-like stem cells (Oct-4A, Sca-1, Nanog), SSCs (Oct-4), and proliferation (Pcna) specific transcripts were upregulated on FSH treatment. Stem cells expressed FSHR and were stimulated by FSH, and Fshr3 was the predominant transcript maximally modulated by FSH. Nuclear OCT-4 and SCA-1 (stem cell antigen 1) positive VSELs are the most primitive stem cells in testis, and FSH stimulates them to undergo asymmetric cell division including self-renewal and give rise to SSCs, which in turn proliferate rapidly and undergo clonal expansion and further differentiation.