Background: The Ste-20 family kinase Hippo restricts cell proliferation and promotes apoptosis for proper organ development in Drosophila. In C. elegans, Hippo homolog also regulates longevity. The mammalian Ste20-like protein kinase, Mst1, plays a role in apoptosis induced by various types of apoptotic stress. Mst1 also regulates peripheral naive T cell trafficking and proliferation in mice. However, its functions in mammals are not fully understood. Methodology/Principal Findings: Here, we report that the Mst1-FoxO signaling pathway plays a crucial role in survival, but not apoptosis, of naive T cells. In Mst1(-/-) mice, peripheral T cells showed impaired FoxO1/3 activation and decreased FoxO protein levels. Consistently, the FoxO targets, Sod2 and catalase, were significantly down-regulated in Mst1(-/-) T cells, thereby resulting in elevated levels of intracellular reactive oxygen species (ROS) and induction of apoptosis. Expression of constitutively active FoxO3a restored Mst1(-/-) T cell survival. Crossing Mst1 transgenic mice (Mst1 Tg) with Mst1(-/-) mice reduced ROS levels and restored normal numbers of peripheral naive T cells in Mst1 Tg; Mst1(-/-) progeny. Interestingly, peripheral T cells from Mst1(-/-) mice were hypersensitive to gamma-irradiation and paraquat-induced oxidative stresses, whereas those from Mst1 Tg mice were resistant. Conclusions/Significance: These data support the hypothesis that tolerance to increased levels of intracellular ROS provided by the Mst1-FoxOs signaling pathway is crucial for the maintenance of naive T cell homeostasis in the periphery.