The bone morphogenetic proteins (BMPs), which are involved in bone formation and repair, play an important role in tissue engineering. For example, BMP-9 and BMP-2, which are members of different BMP subfamilies, are osteoinductive factors. However, several studies have recently shown that BMP-9 is more osteogenic than BMP-2. We have previously shown that fetal bovine serum (FBS) strongly enhances the osteoblast differentiation of murine preosteoblasts (MC3T3-E1) to BMP-9 but not to BMP-2. This effect is mimicked by IGF-2, which primarily activates the PI3K/Akt pathway, but how Akt phosphorylation sites are implicated in such differentiation is unclear. The effects of BMP-9 and BMP-2 with or without FBS or IGF-2 on Akt phosphorylation sites and subsequent osteoblastic differentiation were determined, respectively, by western blot analysis and alkaline phosphatase activity measurements. The involvement of phosphorylated Akt at Thr308 and/or Ser473 on BMP-mediated osteoblast differentiation was further studied using specific inhibitors. In MC3T3-E1 incubated with or without FBS, BMP-9 and BMP-2 activate Akt on Ser473 and Thr308 very differently in a time and dose-dependent manner. Using inhibitors specific to each Akt phosphorylation site, we showed that both Ser473 and Thr308 must be phosphorylated for BMP-9 and/or IGF-2-induced osteoblast differentiation, whereas BMP-2 requires phosphorylation of only Ser473. Furthermore, cells stimulated with BMP-2 in the presence of FBS require the phosphorylation of Akt at Ser473 and the dephosphorylation of Akt at Thr308 to increase the osteoblast differentiation with alkaline phosphatase activity similar to that of BMP-9 plus FBS. These results provide a better understanding into how BMP-9 induces osteoblast differentiation and its synergy with IGF-2 at the signaling level. This knowledge is essential for preparing the serum-free osteogenic media required for bone tissue engineering or developing growth factor delivery systems to improve bone formation.