• First comprehensive multi-omics dissection of chromatin structure dynamics during zebrafish HSPC development, including Hi-C, RNA-seq, ATAC-seq, and ChIP-seq.
• Nascent HSPCs exhibit loose chromatin conformation with obscure hierarchical structures, which reorganize during maturation.
• PU.1 is identified as a key transcription factor mediating promoter-involved chromatin loops and regulating HSPC gene expression.
• Findings provide new insights into epigenetic regulation of vertebrate HSPC fate decision, with implications for improving in vitro HSPC generation.