Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extraembryonic annexes. A rare population of cells resembling 2-cell stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like-cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here, we identify intermediate cellular states and molecular determinants during the emergence of 2-cell-like-cells. By deploying a quantitative single cell expression approach, we identified an intermediate population characterised by the expression of the transcription factor ZSCAN4 as precursor of 2-cell-like-cells. Using an siRNA screening, we uncovered novel epigenetic regulators of 2-cell-like-cell emergence, including the non-canonical PRC1 complex PRC1.6 and Ep400/Tip60. Our data shed light on the mechanisms underlying the exit from the ES cell state towards the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.

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