Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.