Flk-1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) are multipotent cells with extensive proliferation ability and immunomodulatory function. On the basis of our previous work showing their potential application in tissue engineering and immune diseases, we designed a preclinical and Phase I trial to evaluate the feasibility and safety of intravenous infusion of these cells after ex vivo expansion. Rhesus monkey and human Flk-1(+)CD31(-)CD34(-) MSCs were isolated from bone marrow and passaged a maximum of six passages. Karyotype analysis showed Flk-1(+)CD31(-)CD34(-) MSCs remained normally diploid within six passages of expansion. Expanded cells were transplanted into rhesus monkeys and human volunteers, respectively. During the infusion of these cells, the life signs of the recipients were normal. Laboratory tests for blood, bone marrow, kidney, and liver function were conducted and no significant changes were observed before and after cell infusion. Identification of the sry sequence from the male donor in female recipients showed that allogeneic rhesus Flk- 1(+)CD31(-)CD34(-) MSCs were capable of homing to and establishing residence within the recipients' bone marrow. No significant changes were observed in lymphocyte cell subpopulation before and after infusion. These result showed that Flk-1(+)CD31(-)CD34(-) MSCs have no obvious side effects after intravenous administration, encouraging investigators to perform further studies in stem cell therapy.