There are major mechanistic differences among species in how initial cell fate decisions are made in early embryos. Mouse trophoblast differentiation involves CDX2 binding to TCFAP2 sites in the Oct4 promoter, shutting off its transcription. The bovine and human OCT4 promoters lack these sites, suggesting other mechanisms, a concept that is bolstered by co-immunolocalization of CDX2 and OCT4 in early-stage human embryos. To gain insights into the molecular mechanisms underlying lineage allocation in humans, we derived nine cell lines from single blastomeres of four 8-cell embryos (UCSFB1-9). According to established criteria—marker analysis, teratoma formation and directed differentiation—they were human embryonic stem cells (hESCs). We compared their transcriptomes to lines derived by conventional means from intact blastocysts. The blastomere-derived lines differentially expressed cell cycle and metabolic regulators. Conventional hESCs differentially expressed genes that govern developmental processes, evidence of commitment. Comparative analyses of the two methylomes showed significantly hypomethylation of the UCSFB lines at loci that controlled extraembryonic and embryonic development. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by these data, immunolocalization of EOMES and T showed differential expression among blastomeres of 8-12-cell human embryos. As suggested by the methylation data, the lines formed CDX2-positive progeny that yielded a human trophoblast stem cell line, the first of its kind. These results suggested that the UCSFB lines mirror heterogeneity among blastomeres of early-stage embryos and have features of totipotency. Thus, these hESCs have unique properties that make them novel models of the initial stages of human development and potentially valuable tools for cell-based therapies.