||Hematopoiesis is a conserved blood generation process in vertebrates, consisting of two blood generation waves: the primitive and the definitive hematopoiesis. One unique feature of the definitive wave is the existence of hematopoietic stem/progenitor cells (HSPCs), which are not present in the primitive wave. In zebrafish, the HSCs originate from the ventral wall of the dorsal aorta (VDA), then migrate to an intermediate hematopoietic niche called causal hematopoietic tissue (CHT) where the HSPCs pools are established. Finally they home to the kidney where the adult hematopoiesis is maintained. Although the ontogeny of HSPCs has been studied extensively, the genetic program specifying its migration, colonization and maintenance is still not fully understood. In this study, we isolated a zebrafish mutant, named tangohkz3, from a forward genetic screening in Ethylnitrosourea (ENU)-treated zebrafishes. By characterizing the phenotype of tangohkz3, the mutant exhibited a defect of definitive HSPCs maintenance in the CHT, as a consequence, all the definitive blood lineages including erythroid, myeloid and lymphoid cells were impaired. Cellular study showed that the HSPCs in CHT exhibited mild increase of apoptosis and obvious decrease of proliferation. Positional cloning and RNA rescue experiment revealed that SUMO1 activating enzyme subunit 1 (sae1) gene, which coded a component of the activating enzyme for sumoylation modification, was responsible for the mutant phenotype. The Q273X mutation of sae1 was proved to cause insufficient sumoylation modification both in vivo in zebrafish and in vitro in cell lines. More over, the sae1 gene was found ubiquitously expressed in the fish embryo, and was cell-autonomously required for the HSPCs maintenance in the mutant. Our study demonstrated that sae1 is essential for the HSPCs development during zebrafish fetal hematopoiesis.