In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells, such as Glial Fibrillary Acidic Protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER™;tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 days after stroke, and cultured them in neural stem cell medium containing EGF and bFGF. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knockout mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 days after transplantation. In contrast with neurogenic post-natal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.