After a spermatozoon enters an oocyte, maternal factors accumulated in the oocyte reprogram the genomes of the terminally differentiated oocyte and spermatozoon epigenetically and turn the zygote into a totipotent cell, with the capacity to differentiate into all types of somatic cells in a highly organized manner and generate the entire organism, a feature referred to as totipotency. Differentiation of the first lineage begins after three cleavages, when the early embryo compacts and becomes polarized, followed by segregation of the first lineages-the inner cell mass (ICM) and the trophectoderm (TE). To date, a full understanding of the molecular mechanisms that underlie the establishment of totipotency and the ICM/TE lineage segregation remains unclear. In this review, we discuss recent findings in the mechanism of transcriptional regulation networks and signaling pathways in the first lineage separation in the totipotent mouse embryo.