The bone marrow (BM) microenvironment plays a critical role in malignant cell growth, patient survival and response to chemotherapy in hematologic malignancies. However, the molecular mechanisms of BM stromal cells (BMSCs)-mediated survival of tumor cell remain unclear. In this study, to further evaluate the role of Notch-1 in vivo microenvironment, we investigated the influence of inhibiting Notch-1 pathway by Notch-1 siRNA on Jurkat cells in the co-culture system. We found that Notch-1 signaling in Jurkat cells was further activated by interaction with BMSCs, which inhibited drug-induced apoptosis in Jurkat cells. Notch-1 siRNA down-regulated Notch-1 and restored drug-induced apoptosis in co-cultured Jurkat cells. The possible mechanism of restoration of sensitivity to chemotherapy could be associated with repressed Akt signaling. The results indicated that Notch-1 may be a potential mechanism of BMSCs involvement in the protection of hematopoietic malignant cells from drug-induced apoptosis.