Bone marrow-derived cells have important roles in cancer development and progression. Our previous studies demonstrated that murine bone marrow-derived myofibroblasts (BMFs) enhanced tumor growth. In this study, we investigated the mechanisms of BMF actions. We found that co-injection of BMFs with gastric cancer cells markedly promoted tumorigenesis. Co-cultured BMFs or BMF-conditioned medium (BMF-CM) induced the formation of spheres, which expressed stem cell signatures and exhibited features of self-renewal, epithelial-to-mesenchymal transition and tumor initiation. Furthermore, CD44+ fractions in spheres were able to initiate tumorigenesis and re-establish tumors in serially passaged xenografts. In co-culture systems, BMFs secreted high levels of murine interleukin-6 (IL-6) and hepatocyte growth factor (HGF), whereas cancer cells produced high level of transformation growth factor-β1 (TGF-β1). BMF-CM and IL-6 activated BMFs to produce mHGF, which activated signal transducer and activator of transcription 3 (STAT3) and upregulated TGF-β1 in human cancer cells. In return, cancer cell-CM stimulated BMFs to produce IL-6, which was inhibited by anti-TGF-β1 neutralizing antibody. Blockade of HGF/Met, Janus kinase 2 (JAK2)/STAT3 and TGF-β1 signaling by specific inhibitors inhibited BMF-induced sphere formation. STAT3 knockdown in cancer cells also inhibited BMF-induced sphere formation and tumorigenesis. Moreover, TGF-β1 overexpression in cancer cells was co-related with IL-6 and HGF overexpression in stromal cells in human gastric cancer tissues. Our results show that BMF-derived IL-6/HGF and cancer cell-derived TGF-β1 mediate the interactions between BMFs and gastric cancer cells, which regulate cancer stemness and promote tumorigenesis. Targeting inhibition of the interactions between BMFs and cancer cells may be a new strategy for cancer therapy.