The sphingosine 1-phosphate receptor 1 (S1P₁) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P₁ internalization is necessary for this effect. We characterize a knockin mouse (S1p1r^S5A/S5A) in which the C-terminal serine-rich S1P₁ motif, which is important for S1P₁ internalization but dispensable for S1P₁ signaling, is mutated. T cells expressing the mutant S1P₁ showed delayed S1P₁ internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P₁ agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P₁ expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P₁ on T cells is a primary determinant of lymphocyte egress kinetics in vivo.