The study investigates the spatial localization of quiescent hematopoietic stem cells (HSCs) in the bone marrow (BM) and identifies arteriolar niches as critical for maintaining HSC quiescence. Using a novel approach combining whole-mount confocal immunofluorescence imaging and computational modeling, the authors show that quiescent HSCs are preferentially associated with small arterioles, which are distinct from sinusoidal endothelial cells and sinusoid-associated LepR+ cells. These arterioles are surrounded by rare NG2+ pericytes. Pharmacological or genetic activation of HSC cell cycle alters the distribution of HSCs from NG2+ peri-arteriolar niches to LepR+ peri-sinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in the BM. These findings indicate that arteriolar niches are indispensable for maintaining HSC quiescence.The study investigates the spatial localization of quiescent hematopoietic stem cells (HSCs) in the bone marrow (BM) and identifies arteriolar niches as critical for maintaining HSC quiescence. Using a novel approach combining whole-mount confocal immunofluorescence imaging and computational modeling, the authors show that quiescent HSCs are preferentially associated with small arterioles, which are distinct from sinusoidal endothelial cells and sinusoid-associated LepR+ cells. These arterioles are surrounded by rare NG2+ pericytes. Pharmacological or genetic activation of HSC cell cycle alters the distribution of HSCs from NG2+ peri-arteriolar niches to LepR+ peri-sinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in the BM. These findings indicate that arteriolar niches are indispensable for maintaining HSC quiescence.