Mesenchymal stem cells (MSCs) have been reported to be effective against refractory acute graft-versus-host disease (GVHD) following stem cell transplantation because of their immunosuppressive properties. The cases of two pediatric patients who underwent MSC infusions against grade III steroid-refractory acute GVHD that developed after cord blood transplantation (CBT) are reported. One patient was a 4-year-old boy with juvenile myelomonocytic leukemia who developed grade III GVHD involving the liver and gut. His GVHD subsided markedly after the MSC infusion. The other patient was a 6-year-old girl with acute myeloid leukemia who developed grade III GVHD involving the gut. MSC infusion was not effective, but her condition improved gradually with the subsequent administration of conventional immunosuppressive drugs. These two are considered to be the first pediatric patients treated with MSCs in Japan.