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Summary Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals., Graphical Abstract, Highlights • Karyotypically normal pluripotent stem cells derived from single human ICM cells • Expanded lines retain anticipated molecular features of naive embryonic stem cells • Consistency between ICM-derived and in vitro reset naive human pluripotent stem cells • Presence of KLF17 protein in human ICM and naive stem cells, In this paper, Nichols, Smith, and colleagues show that stem cells can be derived from human embryos by dissociation of the inner cell mass into single cells and culture in conditions that suppress further differentiation. Molecular profiling indicates a more naive status than previously observed for human embryonic stem cell lines.