Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet this clinical demand, a fast MSC expansion method is required. In the present study, we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors, including stem cell factor and interleukin-3 and -6. After 8 days of culture, total cell numbers, Stro-1(+)CD44(+)CD34(-) MSCs, and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-, 29-, and 8-fold, respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation, including osteocalcin (osteogenesis), type II collagen (chondrogenesis), and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis), were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs.