Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.