Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow (BM) mesenchymal stromal cells (BM-MSC). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSC revealed that co-culture with leukemia cells upregulated the transcription of genes associated with NF-κB signaling. Moreover, primary BM-MSC from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of IκBα in BM-MSC markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemo-protection. Mechanistic in vitro studies revealed that the interaction between VCAM-1 and VLA-4 played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSC and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.