Chronic myeloid leukemia (CML) is characterized by abnormal proliferation of myeloid cells which when untreated leads to bone marrow failure. Imatinib mesylate (IM) is the first line of therapy for treatment of CML and results in remission in most cases. However, a significant percentage of patients develop chemoresistance to IM, which might be due to the presence of chemoresistant cells in the bone marrow. In the current study, we explored the role of cell-cell interaction of CML cells with the bone marrow stromal cells in the development of chemoresistance in CML. We found that the stromal cells offered long-term chemoprotection to the CML cells from the apoptotic effect of IM. These stroma interacting CML cells were maintained in a non-proliferative stage and had increased ERK1/2 and SMAD1/8 phosphorylation levels. Prolonged interaction of CML cells with the stromal cells in the presence of IM resulted in the acquisition of stroma-free chemoresistance to IM treatment. However, inhibition of actin cytoskeleton, ERK1/2 and SMAD signaling abrogated the chemoresistance acquisition and sensitized the chemoresistant CML cells to IM induced apoptosis.