Soluble factors produced by human marrow stroma or the murine marrow derived M2-10B4 cell line support ex vivo maintenance for 5-8 weeks of 50% of human long-term culture initiating cells (LTC-IC). As the AFT024 cell line supports LTC-IC cultured in contact conditions better than M2-10B4 feeders, we evaluated LTC-IC support in non-contact conditions above AFT024 feeders. We show that only 15% of LTC-IC were maintained for 5 weeks in AFT024 non-contact cultures (n=6, P<0.05). As AFT024-conditioned media added to M2-10B4 non-contact cultures did not inhibit LTC-IC maintenance, AFT024 cells do not secrete factors that inhibit LTC-IC growth. We next characterized heparan sulfate glycosaminoglycans (HS-GAGs) and cytokines produced by AFT024 cells, which are both required for LTC-IC maintenance in M2-10B4 non-contact cultures. The size and extent of O-sulfation of HS-GAGs in AFT024 and M2-10B4 conditioned medium were similar, indicating that absence of hematopoietic specific HS-GAGs is not responsible for the lack of hematopoietic in AFT024 non-contact cultures. Levels of 13 different cytokines secreted in AFT024- and M2-10B4-conditioned medium were similar. However, addition of human SCF, G-CSF, GM-CSF, LIF, MIP-1alpha and IL-6 in concentrations found in human marrow stroma-conditioned medium to AFT024 non-contact cultures increased LTC-IC-maintenance to 72% at 5 weeks. These cytokines improved LTC-IC maintenance in part through interaction with the progenitors and in part, through interaction with the AFT024 feeder. Thus, although LTC-IC maintenance is poor in AFT024 non-contact cultures, addition of human cytokines enhances LTC-IC maintenance in part through indirect effects on the AFT024 feeder. Characterization of known or novel growth factors secreted by AFT024 cells before and after cytokine stimulation may lead to the identification of cytokines that support growth of human hematopoietic stem cells.