Hindlimb unloading, as a simulation of microgravity, decreases the osteogenic potential of mesenchymal stem cells (MSCs) from hindlimb femur of rat. We simulated the microgravity by 28-day of hindlimb unloading for male Sprague-Dawley rat, and performed intramuscular injection of BMP-2 and FGF2 at a given interval during hindlimb unloading. Then, the bone marrow (BM) was collected from hindlimb femur of rat. MSCs were isolated from BM, cultured for four passages, and then induced for osteogenesis. The results revealed that the hindlimb unloading decreased the osteogenic potential of MSCs and also the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Hindlimb unloading for 28 days resulted in the decrease of vinculin-containing focal adhesion in MSCs. During hindlimb unloading, the interval intramuscular injection of BMP-2 or FGF2 alone could increase the osteogenic potential of MSCs and the expression of osteoblast gene marker mRNA. However, the effect of BMP-2 or FGF2 injection alone was significantly lower than that of combination injection of both factors. The further examination showed that the intramuscular injection of BMP-2 promoted the expression of Runx2 mRNA and that the intramuscular injection of FGF2 increased the phosphorylation of ERK and Runx2. Nevertheless, the intramuscular injection of any factor could not increase the formation of vinculin-containing focal adhesions in MSCs. This suggests that BMP-2 should increase the expression of Runx2, and that the activation of Runx2 should be promoted by the FGF2 signaling pathway which activated ERK/Runx2. The activation of this signaling pathway should not lie on the formation of vinculin-containing focal adhesions.