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Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2–4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6–8 h after egg laying (predominantly stage 10–11), to capture a midpoint in embryonic …