The purpose of this study is to investigate whether hematopoiesis capacity, immunomodulatory effect and ex vivo expansion potential of mesenchymal stem cells (MSCs) are affected by cryopreservation. Chronic myeloid leukemia (CML) patients' bone marrow MSCs cryopreserved for 3 months, 6 months, and 1 year were thawed and analyzed. Cryopreserved CML-MSCs have more than 90% viability. Cell-doubling time of cryopreserved CML-MSCs is 42 to 54 hours. Cells have been expanded in culture for more than 30 passages. Under suitable conditions, cryopreserved CML-MSCs have the ability of multiple lineages differentiation, including bone, endothelial, fat and nerve. Furthermore, cryopreserved CML-MSCs express hematopoietic cytokines, and possess hematopoietic supportive ability. The growth of normal long-term culture-initiating cell (LTC-IC) on CML-MSCs (including noncryopreserved and cryopreserved CML-MSCs) was similar to that of normal derived MSCs. Cryopreserved CML-MSCs did not express costimulatory molecules CD40, CD80, and CD86. They can inhibit T lymphocyte proliferation induced by mitogens. The immunosuppressive effect of cryopreserved CML-MSCs on T-cell proliferation was dose dependent. These findings indicate that cryopreserved CML derived MSCs may be a useful tool for clinical application.