To ensure lifelong immunocompetency, naive and memory T cells must be adequately maintained in the peripheral lymphoid tissues. Homeostatic maintenance of T cells is controlled by tonic signaling through T cell antigen receptors and common chain cytokine receptors. In this study, we identify the highly expressed microRNA miR-191 as a key regulator of naive, memory, and regulatory T cell homeostasis. Conditional deletion of miR-191 using LckCre resulted in preferential loss of peripheral CD4(+) regulatory T cells as well as naive and memory CD8(+) T cells. This preferential loss stemmed from reduced survival following deficient cytokine signaling and STAT5 activation. Mechanistically, insulin receptor substrate 1 (Irs1) is a direct target of miR-191, and dysregulated IRS1 expression antagonizes STAT5 activation. Our study identifies a novel role for microRNAs in fine-tuning immune homeostasis and thereby maintaining the lymphocyte reservoir necessary to mount productive immune responses.