In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.