Impaired cell migration has been demonstrated in T cell acute lymphoblastic leukemia (T-ALL) cells upon calcineurin inactivation, among other phenotypic traits including increased apoptosis, inhibition of cell proliferation, and ultimately inhibition of leukemia-initiating cell (LIC) activity. Herein we demonstrate that the chemokine receptor CXCR4 is essential to the LIC activity of T-ALL leukemic cells both in NOTCH-induced mouse T-ALL and human T-ALL xenograft models. We further demonstrate that calcineurin regulates CXCR4 cell-surface expression in a cortactin-dependent manner, a mechanism essential to the migratory properties of T-ALL cells. Because 20%–25% of pediatric and over 50% of adult patients with T-ALL do not achieve complete remission and relapse, our results call for clinical trials incorporating CXCR4 antagonists in T-ALL treatment.