Here we report that a chemical cocktail (LCDM: hLIF, CHIR99021, DiM and MiH) previously used for extended potential pluripotent stem cells enables the de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from bovine embryos and retain developmental potency to differentiate into mature trophoblast cells.