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Day 3 of EVT cell differentiation represented a striking transition point and was readily distinguished from stem state and days 6 and 8 of EVT cell differentiation. Analysis of scRNA-seq led to the identification of several unique cell populations, trophoblast cell developmental state-specific regulons, and trajectories. We elucidated functional roles of key regulators of EVT cell development: cyclin B1, CCAAT\/enhancer-binding protein beta, and A Disintegrin And Metalloproteinase (ADAM) metallopeptidase with thrombospondin type 1 motif 20. Collectively, we have defined developmental landmarks and transitional cell populations during EVT cell differentiation. 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