Only a small number of T cells generated in the thymus each day are selected to replenish the peripheral T cell pool. Much is known about thymic selection; however, little is known of the mechanisms regulating medullary maturation and the release of mature T cells into the blood. Here we demonstrate a rapid acceleration of medullary thymocyte phenotypic maturation through loss of CD69 induced by sphingosine 1-phosphate (S1P) receptor agonist. Low nanomolar agonist concentrations selectively induce changes in CD69(int) CD62L(high) single positive T cells, resulting in down-modulation of CD69 within 2 h. While CD69 loss is accelerated, egress of mature T cells into blood is inhibited >95% within 2 h. Both processes exhibit parallel sensitivities and dose-responses. Together, these data reveal a potent means for rapidly regulating thymic export where S1P receptor agonism alters both phenotypic maturation and egress of thymocytes into blood during late thymic maturation. The S1P system is now shown to acutely regulate both thymic and lymph node egress. Inhibition of lymphocyte egress from thymus and lymph node can contribute synergistically to clinically useful immunosupression by disrupting recirculation of peripheral T cells.