Abstract Niche-derived growth factors (GFs) are essential for establishing and maintaining mammalian spermatogonial stem cells (SSCs) in the adult testis. In culture, however, optimal conditions for expansion of pure populations of self-renewing SSCs remain elusive. We recently found that mouse SSCs possess unexpectedly robust mechanisms to counter-regulate GF signaling by inducing expression of negative feedback regulators (NFRs), including members of the Spry and Dusp gene families. Abrogation of specific NFRs in SSCs led to increase ERK signaling, decreased expression of stem cell-associated genes in vitro and loss of stem cell activity upon transplantation, suggesting that excessive GF-dependent signaling is detrimental to SSC function and may favor differentiation. The cellular alterations that occur following perturbation of NFRs are unknown. However, our data imply that SSCs are programmed to limit ERK signaling within a narrow physiological range. This proposal addresses the mechanisms by which NFRs restrict GF signaling in SSCs, prevent unscheduled differentiation, and enable long-term propagation of SSC clones. Using genetic mouse models, SSC culture, transplantation analysis, and a newly-developed real-time biosensor for ERK activity, we address the following questions: (1) Which NFRs are required for SSC self-renewal? (2) How does ablation of NFRs result in loss of stem cell activity? (3) How do NFRs control the dynamic intracellular signaling cascades downstream of GF receptors? And (4) At which nodes in the ERK pathway do NFRs exert their actions? In addition to providing a rational basis to improve SSC culture systems, these studies will also identify novel adult SSC markers and shed light on mechanisms by which cross-talk between the niche and SSCs balances self-renewal and differentiation in vivo.