Background: Previous studies have shown that mesenchymal stem cells (MSCs) are an important source of somatic stem cells for regenerative medicine. Therefore, the establishment of efficient MSC culture methods to increase their therapeutic value is essential. Here, we focused on cytokines as accelerators of cell proliferation. Methods: We evaluated the effect of the supernatant—obtained from a co-culture of bone marrow-derived MSCs (BMSCs) and myeloid cells—on the proliferation of BMSCs. We then screened for cytokines present in high concentrations in the medium. Next, we added the identified cytokines to the culture medium in various combinations, and cultured BMSCs in this medium to evaluate the effects of cytokines on their proliferation and differentiation potential. Results: The medium obtained from the co-culture of BMSCs with myeloid cells stimulated BMSC proliferation. Nineteen cytokines were present in high concentrations in the medium. Out of the 19 cytokines, MIG and I309 stimulated BMSC proliferation. Furthermore, the culture medium supplemented with MIG and I309 maintained the differentiation potential of cultured BMSCs. Conclusions: MIG and I309 stimulate BMSC proliferation while maintaining their differentiation potential. These results may contribute to the establishment of more efficient MSC culture methods than those currently being used.