We have previously demonstrated in acute myocardial infarctions that human umbilical cord blood mononuclear cells (HUCBCs), which contain hematopoietic, endothelial, and mesenchymal stem cells, reduce acute myocardial infarction size by ≥50% and preserve LV contractility. We hypothesize that the beneficial effects of HUCBCs are due to secretion of biologically active factors that activate in cardiac endothelial cells and myocytes the cell survival protein Akt. We determined by protein microarrays the growth factors and anti-inflammatory cytokines secreted by HUCBCs into culture media during 12 h of hypoxia (1% O2). We then determined by Western blots the effects of cell-free media from hypoxic-conditioned HUCBCs (HUCM) on activation of the cell survival protein Akt in human coronary artery endothelial cells and cardiac myocytes in culture during 24 h of 1% O2. We also determined in separate experiments endothelial cell and myocyte apoptosis by caspase-3 and Annexin V. In the present experiments, HUCBCs secreted multiple growth factors, anti-inflammatory cytokines, and inhibitors of metalloproteinase during normoxia and hypoxia. Human cord blood cells increased the concentration in culture media of angiopoietin, hepatocyte growth factor, interleukin-4, insulin-like growth factor, placental growth factor, vascular endothelial cell growth factor, angiogenin, and stem cell factor by 100 to >10,000% during 12 h of 1% O2 (p<0.001). HUCM, which contained these biological factors, significantly increased Akt phosphorylation/activation in coronary artery endothelial cells and cardiac myocytes subjected to 24 h of 1% O2 by more than 60% (p<0.05) and increased the antiapoptotic protein Bcl-2 expression by 34-50% in comparison with endothelial cells and myocytes treated without HUCM in 1% O2(p<0.05). HUCM also significantly decreased caspase-3 activity and decreased hypoxic endothelial cell and cardiac myocyte apoptosis by more than 40% in comparison with cells cultured without HUCM (p<0.05). Inhibition of Akt activation in endothelial cells and myocytes by the sensitive and specific antagonist API-1 during 24 h of hypoxia nearly completely prevented the beneficial effects of HUCM on inhibiting caspase-3 activity and apoptosis. We conclude that HUCBCs secrete biologically active factors during hypoxia that activate survival proteins in endothelial cells and myocytes that significantly limit apoptosis.