Despite the well-described discrepancy between some of the macroautophagy/autophagy-related genes (ATGs) in the regulation of hematopoiesis, the varying essentiality of core ATGs in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish (Danio rerio) to compare the function of six core atgs from the core autophagy machineries, which included atg13, beclin1 (becn1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via CRISPR-Cas9 ribonucleoprotein targeting. Zebrafish embryos with various atg mutations showed autophagic deficiency throughout the body, including hematopoietic cells. The atgs mutations unsurprisingly caused distinctive hematopoietic abnormalities in zebrafish. Notably, becn1 or atg9a mutation resulted in hematopoietic stem cells (HSCs) expansion during the development of the embryo into a larva, which can be attributed to the proteomic changes in metabolism, HSCs regulators, and apoptosis. Besides, atg3 mutation lowered the leukocytes in developing zebrafish embryos. Intriguingly, a synergistic effect on HSCs expansion was identified in atg13+becn1 and atg9a+atg2a or atg3 double mutations, in which atg13 mutation and atg2a or atg3 mutation exacerbated and mitigated the HSCs expansion in becn1 and atg9a mutations, respectively. In addition, the myeloid cell type-specific effects of various atgs were also determined between neutrophils and macrophages. Of these, a skewed ratio of neutrophils versus macrophages was found in atg13 mutation, while both of them were reduced in atg3 mutation. These findings demonstrated the distinct roles of atgs and their interplays in zebrafish definitive hematopoiesis, thereby suggested that the vertebrate definitive hematopoiesis is regulated in an atgs-dependent manner.