The ability of embryonic stem cells (ESCs) to self-renew and differentiate into a wide range of cell types has encouraged researchers to attempt to isolate ESCs from embryos of domestic species for the past two decades. Success has been limited. The aim of the current study was to investigate whether colonies derived from inner cell masses (ICMs) of bovine blastocysts expressed the same markers of pluripotency and candidate genes representing the various signaling pathways as those found in human or mouse ESCs. The ability of selected cytokines to maintain the major transcription factors associated with pluripotency (NANOG, POU5F1 and SOX2) in the ICM explants was also tested. The results of the study showed that the three major transcription factors (NANOG, POU5F1 and SOX2) were expressed initially in culture but were lost with continued culture and passaging. Markers of differentiation (BMP4, HNF4, NCAM, and CDX2) were also expressed in the initial days of culture. The candidate genes representing the various signaling pathways were expressed in the initial days of culture as well as in subsequent passages. Noggin, a cytokine inhibiting the BMP4 pathway successfully up-regulated the relative expression of NANOG in the ICM explants with respect to controls. The results indicate that signaling pathways associated with regulating pluripotency are expressed in ICM explants and that with cytokine supplementation pluripotency may be maintained. An alternate approach in which differentiating cells in the primary colonies were selectively ablated to eradicate cells secreting pro-differentiation signals was tested. Bovine embryos that carried the hygromycin resistance gene driven by the NANOG promoter were generated by SCNT. Any pluripotent colonies generated from these embryos should survive in the presence of hygromycin. When cultured in the presence of Noggin and hygromycin, colonies were generated; however they failed to proliferate on passaging. This suggests that the culture conditions were not optimal for the NANOG promoter to remain active over extended culture.