Recently, we have manipulated endogenous neural stem/progenitor cells (NSCs) in situ in the adult mouse to undergo neurogenesis and anatomic circuit re-formation de novo in the neocortex, where it does not normally occur, by using a highly targeted brain injury model. However, how the NSCs respond to injury in the adult mouse brain is poorly understood. While studying the molecular mechanisms that regulate NSC fates after brain injury, it is important to develop a strategy to identify NSCs in niches and isolate them directly from fresh tissue after brain injury. Here we report that we directly isolated NSCs from adult brains after traumatic brain injury by genetically labeling NSCs with EGFP combined with fluorescence-activated cell sorting (FACS) technique without an intervening cell culture and with high concentrations of growth factors. The isolated EGFP-positive cells can self-renew and have the potential to differentiate into both neurons and glia in vitro, confirming that the FACS-sorted EGFP-positive cells are NSCs. This unique approach provides a useful tool to isolate large amounts of endogenous NSCs in situ for identifying the critical molecules that regulate fate decision and neurogenesis in the adult brain after injury.