Transplantation of stem cells is a potential therapeutic strategy for stroke damage. The survival, migration, and differentiation of transplanted human embryonic neural stem cells in the acute post-ischemic environment were characterized and endogenous nestin expression after transplantation was investigated. Human embryonic neural stem cells obtained from the temporal lobe cortex were cultured and labeled with fluorescent 1,1'-dioctadecy-6,6'-di (4-sulfopheyl)-3,3,3',3'-tetramethylindocarbocyanin (DiI) in vitro. Labeled cells were transplanted into cortical peri-infarction zones of adult rats 24 h after permanent middle cerebral artery occlusion. Survival, migration, and differentiation of grafted cells were quantified in immunofluorescence-stained sections from rats sacrificed at 7, 14, and 28 days after transplantation. Endogenous nestin-positive cells in the cortical peri-infarction zone were counted at serial time points. The cells transplanted into the cortical peri-infarction zone displayed the morphology of living cells and became widely located around the ischemic area. Moreover, some of the transplanted cells expressed nestin, GFAP, or NeuN in the peri-infarction zone. Furthermore, compared with the control group, endogenous nestin-positive cells in the peri-infarction zone had increased significantly 7 days after cell transplantation. These results confirm the survival, migration, and differentiation of transplanted cells in the acute post-ischemic environment and enhanced endogenous nestin expression within a brief time window. These findings indicate that transplantation of neural stem cells into the peri-infarction zone may be performed as early as 24 h after ischemia.