Background: hESCs-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods: Herein, hESCs-MSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN , VIMENTIN , CD10 , CD105 , and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results: MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. The hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion: Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.