The bovine blastocyst hatches 8-9 days after fertilization and this is followed by several days of pre-implantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e. the blastocoel fluid gradually alters to become the primitive yolk sac fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive yolk sac fluid and the embryonic cells during early slightly later cell differentiation in the developing bovine embryo. In vitro produced day six embryos were transferred into a recipient heifer and after seven days of further in vivo culture, ovoid and elongated day 13 embryos were recovered by flushing both uterine horns following slaughter. The primitive yolk sac fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano high-performance liquid chromatography (HPLC), tandem mass spectrometry, and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term- and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a significant contribution to the current knowledge of bovine pre-implantation development.