This study was aimed to develop methods for isolation, culture and characterization of embryonic cell lines from in vitro produced bovine blastocysts. Inner cell masses arising from blastocysts were isolated by immunosurgery onto mitomocin-C-inactivated mouse embryonic fibroblast (MEF). After 10 to 15 days of culture the primary cell colonies were disaggregated, seeded in a new MEF, and cultured for 3 to 6 days up to form new colonies. The primary cell colonies, passage 2, passage 3 and post-thawed colonies expressed pluripotency markers such us SSEA-4, TRA-1-60 and Oct4 and were alkaline phosphatase positive. More research is needed to confirm pluripotency and selfrenew stage within the obtained embryonic stem-like cells (ES-like).