Neural stem cell specification depends on antero-posterior (AP) and dorso-ventral (DV) information provided during development. In the present study we identified similar neural stem cell (NSC) populations along the AP axis of the mouse central nervous system: the ‘early’ NSCs responsive to fibroblast growth factor-2 and the ‘late’ NSCs responsive to epidermal growth factor (EGF). Gene expression analysis shows that AP and DV transcription factor code is not preserved in NSCs in culture. Neurospheres generated with EGF from different regions showed Emx2, En2 and Krox20 expression beyond their corresponding AP restricted areas (telencephalon, mesencephalon and rhomboencephalon, respectively). Hox genes were rarely expressed. DV markers such as Pax7 and Dbx1 were not expressed in neurosphere cells, whereas Pax6 and Nkx2.1 were highly expressed independently of the NSC source region. In general, this pattern was found under different culture conditions. We propose that signals surrounding NSCs determine their positional identity gene expression code, which may be relevant to establish their definitive fate.