HepG2 cell line has a characteristic of visceral endoderm that was known as a source of signals during embryo development. This cell line secretes soluble signalling factors that are regarded as equivalent to the signals from visceral endoderm. The HepG2-cell-secreted signalling factors induce mesoderm effectively from embryonic stem cells preventing ectoderm development. Therefore elucidation of HepG2 cell-secreted factors will not only help us to understand complex signalling system of early embryo development in vivo but it will also give us a noble way of generating embryo stem cell derived final lineage cell source that can be used for skeletal tissue regeneration. To achieve identification of the active factors in the HepG2 CM, we adapted the HepG2 cell line in serum free culture and used proteomics method to identify key components in the HepG2 conditioned medium. Two-dimensional gel electrophoresis will separate proteins and the protein spots will be identified by peptide mass fingerprinting using MALDI-MS. However it is not easy to target all the proteins secreted by HepG2 cells since it will come up with numerous possibility of combination of factors. Therefore we compared the proteomes of two visceral endoderm-like cell line conditioned media, namely HepG2 and END-2. Then the common factors can be feasibly identified by DIGE. Once the list of candidate proteins was obtained, those factors are going to be validated using the loss and gain of function method for their mesoderm inducing activity and further osteogenic differentiation activity starting on mESCs. The final step will be the application of the identified molecules to the hESCs and introduce a noble process that can be practically exploited in the clinical skeletal tissue therapy.