Development of hematopoietic stem and progenitor cells (HSPC) is a multi-staged complex process that conserved between zebrafish and mammals; however, the mechanism underlying HSPC development is not fully understood. Chromatin conformation plays important roles in transcriptional regulation and cell fate decision, its dynamic and role in HSPC development is poorly investigated. Here, we performed chromatin structure and multi-omics dissection across different stages of HSPC developmental trajectory in zebrafish. Chromatin organization of zebrafish HSPC resemble mammalian cells with similar hierarchical structure and characteristics. We revealed the multi-scale reorganization of 3D genome and its influence on transcriptional regulation and transition of cell function during HSPC development. Nascent HSPC is featured by loose conformation with obscure structure at all layers. Notably, PU.1 was identified as a potential factor mediating formation of promoter-involved loops and regulating gene expression as well as HSPC function. Our results provided a global view of chromatin structure dynamics associated with development of zebrafish HSPC and discovered key transcription factor involved in HSPC chromatin interactions, which will provide new insights into the epigenetic regulatory mechanisms underlying vertebrate HSPC fate decision.