In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango(hkz5), which shows defects of definitive hematopoiesis. In tango(hkz5) mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango(hkz5) encodes SUMO1-activating enzyme subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango(hkz5) mutation produces a truncated Sae1 protein (ΔSae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.