Updated project metadata. Heterochromatin is established at the 2-cell stage progression during mouse embryonic development. Here, we show that cell cycle arrest controls heterochromatin establishment in the transition from 2-cell-embryo-like (2C-like) cells to pluripotency. Conversion of embryonic stem cells (ESCs) to 2C-like cells is marked by a reorganization of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. We characterized the chromatome of 2C-like cells by genome capture, uncovering a relationship between cell cycle regulation and the establishment of the 2C-like state. Cell cycle arrest in S-G2/M induces 2C-like cells, which in turn accumulate in G2/M after cell fate conversion. Depletion of SMARCAD1, an interactor of cell cycle regulators identified by chromatin proteomics that co-localizes with H3K9me3-heterochromatin, leads to the arrest of embryo development and loss of H3K9me3. Our findings demonstrate that tight control of cell cycle progression is important for the establishment of heterochromatin foci during the transition from 2C-like cells to pluripotency.