Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.