Bone marrow microenvironment is essential for leukemia cells to survive and escape the killing effect of chemotherapeutics. Cancer-associated fibroblasts (CAFs) are the dominant stromal cells in tumor microenvironment (TME), but their role in B-cell acute lymphoblastic leukemia (B-ALL) remains unclear. Here, RT-PCR and Western blotting in bone marrow mononuclear cells revealed higher proportions of CAFs markers α-SMA and FAP in the newly diagnosed and relapsed B-ALL patients. In vitro experiments, bone marrow mesenchymal stem cells (BM-MSCs) acquired a CAFs phenotype after co-culture with leukemia cells, which produced high level of tumor-promoting growth factors and reduced the daunorubicin (DNR)-induced damage to B-ALL cells. As for its mechanism, CAFs activation was mediated by TGF-β up-regulation in the co-culture system, and TGF-β triggered MSCs conversion into CAFs relying on the SDF-1/CXCR4 pathway. Further LY2109761 and AMD3100 effectively decreased the activation of CAFs through inhibiting TGF-β receptor and CXCR4. Comparative experiments with MSCs and transformed CAFs prompted that CAFs had more obvious effect than MSCs on stimulating leukemia progression through accelerating leukemia cell migration and invasion. These results clarified the important role of CAFs in B-ALL progression and the possible mechanisms of CAFs activation in leukemia microenvironment, which might provide a theoretical basis for B-ALL patients to find more effective targeted therapies targeting the bone marrow microenvironment.