The mammalian post-implantation embryo has been vastly investigated at the tissue level.However, molecular characteristics of individual cells are unclear.Here we investigate gene expression patterns in single cells freshly isolated from embryonic day (E) 5.5 and E6.5 mouse embryos.First, single-cell RNA-sequencing (RNA-Seq) data are obtained from 101 cells from 3 embryos, mainly containing epiblast cells,which can be clearly distinguished from extraembryonic cells by the transcriptome data.Many pluripotency markers, including na(i)ve pluripotency markers, are still expressed.Simultaneously, some lineage-specific markers can already be detected.Neuroectoderm and mesendoderm markers are broadly distributed in a heterogeneous pattern.Interestingly, some cells express higher levels of mesendoderm markers and lower levels of Sox3 than other cells, therefore displaying mesendoderm-like characteristics.Then, single-cell high-throughput quantitative real-time PCR experiments are performed on 247 cells, confirming the existence of mesendoderm-like cells and further revealing another type of E6.5 embryos, in which the number of primitive-streak cells double-positive for Oct4 and Gata6 (Oct4+Gata6+) increases.Expression of mesendoderm marker intensifies in these cells.Surprisingly, a subgroup of Oct4+Gata6+ cells enriches endoderm signatures.Collectively, our study provides not only previously unavailable single cell gene expression information but also insights into early cellular decision-making in embryos immediately after implantation.