In spite of advances in the treatment of pediatric acute lymphoblastic leukemia (ALL), a significant number of children with ALL are not cured of their disease. We and others have shown that signaling from the bone marrow microenvironment confers therapeutic resistance, and that the interaction between CXCR4 and stromal cell-derived factor-1 (SDF-1 or CXCL12) is a key mediator of this effect. We demonstrate that ALL cells that upregulate surface CXCR4 in response to chemotherapy treatment are protected from chemotherapy-induced apoptosis when co-cultured with bone marrow stroma. Treatment with the CXCR4 inhibitor plerixafor diminishes stromal protection and confers chemosensitivity. Using xenograft models of high-risk pediatric ALL, plerixafor plus chemotherapy induces significantly decreased leukemic burden, compared to chemotherapy alone. Further, treatment with plerixafor and chemotherapy influences surface expression of CXCR4, VLA-4, and CXCR7 in surviving ALL blasts. Finally, prolonged exposure of ALL blasts to plerixafor leads to a persistent increase in surface CXCR4 expression, along with modulation of surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis, findings that may have implications for therapeutic resistance. Our results suggest that while CXCR4 inhibition may prove useful in ALL, further study is needed to understand the full effects of targeting the leukemic microenvironment.