Self-renewal and differentiation of spermatogonial stem cells (SSCs)provide the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1⁺ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1⁺ germ cell fraction with the Thy1-depleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1⁺ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1⁺ germ cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1⁺ spermatogonial cultures over a 63-day period without affecting total germ cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig cells and select peritubular myoid cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid cells as contributors of the testicular stem cell niche in mammals.