We here investigated whether receptor tyrosine kinase signaling (ckit, flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture, or whether, like in embryonic stem cells, signaling through gp130 is also required. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from human umbilical cord blood (CB) were cultured in the presence of combinations of ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and different stimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of these cytokines, SCF stimulated cell division by itself. Gp130-stimulating cytokines did not give any additional cell division activity. When SCF was included in the cytokine cocktail, progenitor levels (CFC, CAFCw2, CAFCw6) was very similar for all combinations of cytokines tested after this initital 6-day culture. To study more immature progenitors, cells were harvested after 6 days of serum-free culture and cultured for another 6 weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showed that in initial 6 day cultures with FL or gp130-stimulators alone, very little hematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However, combinations of SCF with OSM consistently increased the number of LTC-CAFCw2 and w6 about two-fold. This finding suggests that very immature hematopoietic progenitors were at least maintained in serum-free cultures. To study the most immatore cells, we performed in transplantations into sublethally irradiated NOD/SCID mice. When CB cells were cultured with SCF alone, NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped to about 20% in 6 days (1 in 5500). Similarly, cultures without growth factors or with FL or OSM showed a decrease to less than 10% of the original CRU number. In contrast, the combination of SCF and OSM maintained CRU at about half the original level (1 in 1900). This study shows that signals through ckit and gp130 synergize to maintain CRU level. However, to achieve expansion of human NOD/SCID-repopulating cell numbers, additional signaling pathways need to be stimulated.