Robust and large-scale expansion of umbilical cord blood stem cells in vitro is necessary for widening the usage of transplantation therapies for the treatment of hematological and immune diseases. The lack of understanding of the complex inter-cellular networks regulating stem cell fate in culture explains the low success met so far for the ex vivo expansion of hematopoietic stem cells. The development of a mathematical model of in vitro hematopoiesis coupled with gene expression profiling led to predictions about the secreted factors that play a crucial role in regulating hematopoietic stem cell self-renewal in culture. We tested 18 putative molecules predicted to display effects on primitive progenitor (Long-Term Culture-Initiating Cell; LTC-IC) output, functionally validating three stimulators (VEGF, PDGF, EGF) and three inhibitors (TGFβ, CCL4, CXCL10). Combinatorial studies with the stimulatory molecules showed less-than additive effects, perhaps related to redundant signaling mechanisms. Small molecule-mediated inhibition of the downstream signaling pathways activated by VEGF, PDGF, and EGF led to a decreased expansion of primitive cell compartments, confirming the endogenous activity of these growth factors for the stimulation of blood stem and progenitor cells. Blocking TGFβ-mediated negative feedback signaling in culture enhanced mature cell outputs but had no effect on primitive cell expansion, consistent with the role of TGFβ as a proliferation inhibitor, and underlying the complexity and multi-parametric aspect of the inter-cellular regulatory network. Studies are currently underway to validate the functional activity of VEGF and EGF on in vivo repopulating stem cells (NOD/SCID repopulating cells; SRC) using a clinical-grade, closed-system bioprocess. These results constitute a major step in the functional elucidation of the complex extracellular signaling networks that govern stem cell fate in vitro