Since the discovery of adult neural stem cells, their exact identity is still under discussion. Moreover, the lack of a reproducible procedure to purify neural stem cells prospectively rather than by growing them in vitro has so far precluded their study at the transcriptome level. Here we demonstrate a novel procedure to prospectively isolate neural stem cells from the adult mouse subependymal zone on the basis of their GFAP- and prominin1-expression by fluorescence-activated cell sorting. All self-renewing, multipotent stem cells are contained in this fraction at 70% purity. The stem cell identity of these double-positive cells is further demonstrated in vivo, by using a novel split-Cre-technology for fate mapping. Comparison of the transcriptome of these prospectively isolated neural stem cells to the transcriptomes of other astrocytes or ependymal cells revealed a distinct clustering of the stem cells, highlighting their unique features, as well as profound similarities to the astrocyte as well as ependyma transcriptome. FACS-sorting was used to isolate prospective neural stem, astrocytes and ependymal cells from brain regions of hGFAP eGFP expressing mice. Total RNA from these cells was hybridised on Affymetrix MOE430 2.0 arrays and expression patterns were analysed.