Hypoxia is considered an important factor in the proliferation and stemness of mesenchymal stem cells (MSCs). This study aimed to obtain large numbers of mouse clonal MSCs (mc-MSCs) and evaluate the effect of hypoxia on the proliferation and differentiation of mc-MSCs. mc-MSCs were cultured on a Cytodex 3 microcarrier in a spinner flask for a suspension culture to increase mass productivity. To produce the hypoxic conditions, CoCl2 and Na2SO3 were used in a low glucose DMEM medium. The hypoxia environment was established using 0.5g/L Na2SO3 with 10μM or 100 µM CoCl2 for 24 hours. As a result, the proliferation of mc-MSCs under hypoxic conditions was 1.56 times faster than the control group over seven days. The gene expression of HIF-1a and VEGFA increased 4.62 fold and 2.07 fold, respectively. Furthermore, the gene expression of ALP, RUNX2, COL1A, and osteocalcin increased significantly by 9.55, 1.55, 2.29, and 2.53 times, respectively. In contrast, the expression of adipogenic differentiation markers, such as PPAR-γ and FABP4, decreased. These results show that the hypoxia environment produced by these chemicals in a suspension culture increases the proliferation of mc-MSCs and promotes the osteogenic differentiation of mc-MSCs.