Bone marrow niche cells have been reported to fine-tune hematopoietic stem cell (HSC) stemness via direct interaction or secreted components. Nevertheless, how niche cells con-trol HSC activities remains largely unknown. We previously showed that angiopoietin-like protein 2 (ANGPTL2) can support the ex vivo expansion of HSCs by binding to human leu-kocyte immunoglobulin-like receptor B2. However, how ANGPTL2 from specific niche cell types regulates HSC activities under physiological conditions is still not clear. Herein, we generated an Angptl2-flox/flox transgenic mouse line and conditionally deleted Angptl2 expression in several niche cells, including Cdh5(+) or Tie2(+) endothelial cells, Prx1(+) mesenchymal stem cells, and Pf4(+) megakaryocytes, to evaluate its role in the regulation of HSC fate. Interestingly, we demonstrated that only endothelial cell-derived ANGPTL2 and not ANGPTL2 from other niche cell types plays important roles in supporting repopulation capacity, quiescent status, and niche localization. Mechanistically, ANGPTL2 enhances per-oxisome-proliferator-activated receptor D (PPARD) expression to transactivate G0s2 to sustain the perinuclear localization of nucleolin to prevent HSCs from entering the cell cycle. These findings reveal that endothelial cell-derived ANGPTL2 serves as a critical niche component to maintain HSC stemness, which may benefit the understanding of stem cell biology in bone marrow niches and the development of a unique strategy for the ex vivo expansion of HSCs.