The bone marrow (BM) stromal niche can protect acute lymphoblastic leukemia (ALL) cells against the cytotoxicity of chemotherapeutic agents and is a possible source of relapse. The stromal-derived factor-1 (SDF-1)/CXCR4 axis is a major determinant in the crosstalk between leukemic cells and BM stroma. In this study, we investigated the use of AMD11070, an orally available, small-molecule antagonist of CXCR4, as an ALL-sensitizing agent. This compound effectively blocked stromal-induced migration of human ALL cells in culture and disrupted pre-established adhesion to stroma. To examine how to optimally use this compound in vivo, several combinations with cytotoxic drugs were tested in a stromal co-culture system. The best treatment regimen was then tested in vivo. Mice transplanted with murine Bcr/Abl ALL cells survived significantly longer when treated with a combination of nilotinib and AMD11070. Similarly, immunocompromised mice transplanted with human ALL cells and treated with vincristine and AMD11070 had few circulating leukemic cells, normal spleens and reduced human CD19+ cells in the BM at the termination of the experiment. These results show that combined treatment with AMD11070 may be of significant benefit in eradicating residual leukemia cells at locations where they would otherwise be protected by stroma.