The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM- and epiblast-derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)-like morphology. A total of 104 zona pellucida-enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC-like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC-like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC-like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT-PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC-like morphology. In conclusion, we have established a robust system for derivation of ESC-like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC-like morphology although this relationship is lost during early passages.