Abstract: Bovine ES cells have been isolated but validated bovine ES cell lines have not yet been successfully established. Here, researchers investigated the factors that affect the derivation, proliferation and maintenance of bovine Embryonic Cell (bESC)-like cells including type of feeders (Mouse Embryonic Fibroblasts (MEFs) vs. Bovine Embryonic Fibroblasts (BEFs)), feeder density and inner cell mass isolation procedure. We found that the MEFs work better than BEFs on the process and best at a cell density of 1.25x105 cells/cm2, naturally hatched blastocysts had a short adherence time and high rate of adherence. They were more conducive to bESC-like cell isolation and culture. Culture medium supplemented with IGF promoted proliferation of bESC-like cells but was unfavorable for the maintenance of pluripotency. The findings provide information on the establishment of culture systems for bESC-like cells.