Abstract Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two‐round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient‐rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this “on‐gel” culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on‐gel‐cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA‐seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency‐related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways‐related genes to facilitate the functional maturation required for feto‐maternal interaction. Quantitative PCR analysis of TE cells derived from on‐gel culture further confirmed time‐dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species‐specific strategies for mammalian preimplantation development.