Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.