Autologous human bone marrow stromal cells (hBMSCs) should be expanded in the animal serum-free condition within clinically relevant periods in order to secure safe and effective cell therapy for ischemic stroke. This study was aimed to assess whether the hBMSCs enhance their proliferation capacity and provide beneficial effect in the infarct brain when cultured with platelet lysate (PL) and granulocyte-colony stimulating factor (G-CSF). The hBMSCs were cultured in the fetal calf serum (FCS)-, PL-, or PL/G-CSF-containing medium. Cell growth kinetics was analyzed. The hBMSCs-PL, hBMSC-PL/G-CSF, or vehicle was stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Motor function was assessed for 8 weeks, and the fate of transplanted hBMSCs was examined using immunohistochemistry. As the results, the hBMSCs-PL/G-CSF showed more enhanced proliferation than the hBMSCs-FCS and hBMSCs-PL. Transplantation of hBMSCs expanded with the PL- or PL/G-CSF-containing medium equally promoted functional recovery compared with the vehicle group. Histological analysis revealed that there were no significant differences in their migration, survival, and neural differentiation in the infarct brain between the hBMSCs-PL and hBMSCs-PL/G-CSF. These findings strongly suggest that the combination of PL and G-CSF may accelerate hBMSC expansion and serve safe cell therapy for patients with ischemic stroke at clinically relevant timing.