Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both naïve and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the naïve state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4.