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Notch is long recognized as a signaling molecule important for stem cell self‐renewal and fate determination. Here, we reveal a novel adhesive role of Notch‐ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of  O ‐fucosylglycans on Notch EGF‐like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand‐expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand‐bearing osteoblastic or stromal cells inhibits wild type but not  O ‐fucosylglycan‐deficient HSPC cycling, independent of  RBP‐J   K  ‐mediated canonical Notch signaling. Furthermore, Notch‐ligand neutralizing antibodies induce  RBP‐J   K  ‐independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor–ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on  O ‐fucosylglycans on Notch. S tem  C ells  2015;33:2280–2293

Notch Receptor‐Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention