We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1α (MIP-1α), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1α binding ability. A specific MIP-1α-binding HS oligosaccharide preparation of Mr 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1α binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.