Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft-vs.-host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture-expanded MSCs and HSCs from the same human leukocyte antigen (HLA)-identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 +/- 0.40) x 10(6)/kg (donor's weight) MSCs were successfully expanded from 23.6 +/- 5.96 ml of bone marrow samples. They had normal karyotypes with bi-lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II-IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29-57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.