The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34⁺cells and ex vivo expanded early colony-forming unit–granulocyte-macrophage (CFU-GM)–derived, burst forming unit–erythroid (BFU-E)–derived, and CFU–megakaryocyte (CFU-Meg)–derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor–2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor–1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor–α, Fas ligand, interferon α, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein–1α [MIP-1α], MIP-1β, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein–3 [MCP-3], MCP-4, IL-8, interferon-inducible protein–10, macrophage-derived chemokine [MDC], and platelet factor–4 [PF-4]) to be expressed by CD34⁺ cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor–β1 (TGF-β1), TGF-β2, RANTES, MIP-1α, MIP-1β, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34⁺ cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34⁺ cells; chemo-attract CFU-GM– and CFU-Meg–derived cells as well as other CD34⁺ cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM–, CFU-Meg–, and BFU-E–derived cells. It is concluded that normal human CD34⁺ cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.