The promising cell sources for tissue engineering are bone marrow derived-mesenchymal stem cells (BM-MSCs) that have multiple differentiation potentials. Also, sex hormones act as important elements in bone development and maintenance, the roles of two female sex steroid hormones known as estrogen (17-β estradiol) and progesterone in osteogenic differentiation of human BM-MSCs were studied. For this purpose, human BM-MSCs were treated with a 1×10 M concentration of 17-β estradiol and progesterone separately and simultaneously while the optimum concentrations were obtained by MTT assay. Osteogenic differentiation tests including measurement of ALP enzyme activity, the content of total mineral calcium, mineralized matrix staining by Alizarin Red and Von Kossa solutions, Real-time RT-PCR, and immunofluorescence staining have been carried out on day 7 and 14 of differentiation. To exhibit the morphology of the cells, the BM-MSCs were stained by Acridine Orange (AO) solution. In this research, the result of ALP activity assay, calcium content and Real-time RT-PCR assay and also all tests of differentiation staining have shown that 17-β estradiol has been recognized as an enhancing factor of osteogenic differentiation. Furthermore, MTT assay and AO staining revealed progesterone as a factor that seriously improved the proliferation of human BM-MSCs. Generally, the 17-β estradiol individually or in the presence of progesterone has more effect on BM-MSCs' osteogenic differentiation compared to progesterone alone. In this study, it is indicated that the effect of the 17-β estradiol and progesterone concurrently was the same as individual 17-β estradiol on the differentiation of human BM-MSCs. This article is protected by copyright. All rights reserved.