Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date, it has been challenging to establish pluripotent ESC lines for domestic animals, which could be important for biotechnological applications, such as genetic engineering and SCNT, and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech) with 4 mg/mL BSA for 7 days until they reached the blastocyst stage (Ross et al., 2009 Reproduction 137, 427–437). At that point, the zona pellucida (ZP) was removed using 1 mg/mL Pronase (Sigma, St. Louis, MO), and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts, mechanically isolated ICM, and immunosurgery-derived ICM. In each case, individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20 ng/mL FGF2 and 2.5 μm IWR1 (CTFR) (Wu et al. 2015 Nature 521, 316–321). After 48 h, blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter, the media was changed daily. Outgrowths (after 6–7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632, 10 μm) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF), RNA-seq, and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16, 56.3%), mechanical ICM isolation (7/12, 58.3%), and immunosurgical ICM isolation (7/16, 43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency), but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1, NANOG, SOX2, LIN28B, DNAMT3B, UTF1, SALL4) were expressed (RPKM > 0.4), while trophectoderm markers (CDX2, GATA2, GATA3, FGF4, TFAP2A) and primitive endoderm markers (GATA6, HNF4A) were not expressed (RPKM < 0.4). Finally, bESC lines (n = 2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1, endoderm: FOXA2, and mesoderm: ASM). In conclusion, the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC.