Clec16a maintains definitive hematopoietic stem and progenitor cells via mitophagy

• CLEC16A is essential for arterial formation and definitive hematopoiesis • CLEC16A loss impairs ATG5-dependent mitophagy and elevates mitochondrial ROS • CLEC16A prevents K48-linked ubiquitination and degradation of ATG5 • A CLEC16A-USP8-ATG5 axis maintains mitochondrial homeostasis during EHT CLEC16A is essential for arterial formation and definitive hematopoiesis CLEC16A loss impairs ATG5-dependent mitophagy and elevates mitochondrial ROS CLEC16A prevents K48-linked ubiquitination and degradation of ATG5 A CLEC16A-USP8-ATG5 axis maintains mitochondrial homeostasis during EHT Hematopoietic stem and progenitor cells (HSPCs) arise from hemogenic endothelium via the endothelial-to-hematopoietic transition (EHT), a process requiring precise mitochondrial quality control. Here, we identify Clec16a , an E3 ubiquitin ligase, as a conserved regulator of embryonic HSPC emergence. In zebrafish and HEK293T models, Clec16a is enriched in hemogenic endothelium, and its loss disrupts arterial identity, impairs EHT, and reduces lymphoid, erythroid, and myeloid lineages. Transcriptomic and proteomic analyses show that Clec16a deficiency compromises mitophagy by promoting aberrant K48-linked ubiquitination and proteasomal degradation of ATG5, leading to mitochondrial dysfunction and elevated reactive oxygen species. These findings establish Clec16a as an essential regulator linking ubiquitin signaling, mitophagy, and hematopoietic fate specification. Our study defines a mitophagy-dependent checkpoint that safeguards mitochondrial homeostasis during developmental hematopoiesis and provides insight into the metabolic control of hematopoietic disorders.