O-fucosylglycans on Notch extracellular domain epidermal growth factor (EGF) repeats are critical in modulating Notch signaling by altering the sensitivity of Notch receptors to activation by Notch ligands. Mice lacking Notch O-fucosylglycans display granulo-monocytic myeloproliferation, hematopoietic stem cell dysfunction and aberrant stem cell niche occupancy. The inability of the stem cells/progenitors that lack Notch O-fucosylglycans to transcribe Notch signaling activation is attributed by a loss of effective Notch-ligand interaction. These findings, in conjunction with myeloproliferation identified in mouse models with defective Notch cleavage or ligand endocytosis, reveal emerging new roles of Notch in hematopoietic stem cell biology and myeloid homeostasis.