Endothelial cells can be successfully used to maintain or increase the number of hematopoietic stem cells in vitro. Previously we identified hematopoietic progenitor cell (HPC) expansion or survival benefit induced by IL-1β-, IL-3-, and IL-6-stimulated human umbilical vein endothelial cell (HUVEC) supernatants. In order to identify molecular mechanisms that support hematopoiesis, we examined the time-dependent expression profiles of IL-1β-, IL-3-, and IL-6-stimulated HUVECs via microarray. Here, we present 24 common upregulated elements and three common downregulated elements of IL-1β- and IL-3-stimulated HUVECs, with these factors exhibiting great potential for the observed HPC expansion. Furthermore, metabolic pathway analysis resulted in the identification of nonproteinogenic factors such as prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and determined their HPC expansion potential via delta, methylcellulose, and cobblestone assays. We confirmed PGE(2) and spermine as hematopoietic expansion factors. Furthermore, we identified several factors such as SSAT, extracellular matrix components, microRNA21, and a microvesicle-mediated cross-talk between the endothelium and HPCs that may play a crucial role in determining stem cell fate. Our results suggest that microarray in combination with functional annotations is a convenient method to identify novel factors with great impact on HPC proliferation and differentiation.