NS cells are a homogeneous population of neural stem cells which were previously derived from embryonic stem cells as well as from the fetal and adult brain. Our previous reports have described a 21 day long neuronal differentiation protocol able to reproducibly convert adult SVZ-derived NS (aNS) cells into a population composed of 65% mature neurons and 35% glial cells. Here we have developed a different procedure specifically applicable to ES-derived NS cells in order to fully explore their neurogenic capacity. Differently from the aNS differentiation procedure, optimized neuronal output from ES-derived NS cells requires replating of the cells on appropriate substrates followed by sequential exposure to modified media. In these conditions, ES-derived NS cells differentiate into neurons with a barely appreciable quota of astrocytes and occasional oligodendrocytes. In particular, 21 days after the beginning of the treatment, 85% of the cells has differentiated into molecularly and electrophysiologically mature neurons belonging to the GABAergic lineage. The procedure, which is applicable with no considerable differences to different ES-derived NS cell lines and to NS cells at different passages, opens to the possibility of molecular and biochemical studies on close-to-uniform stem cell derived neurons.