How mammalian neuronal identity is progressively acquired and reinforced during development is not understood. We have previously shown that loss of RP58 (ZNF238, ZBTB18), a BTB/POZ and zinc finger containing transcription factor, in the mouse brain leads to microcephaly, corpus callosum agenesis, cerebellum hypoplasia and that it is required for normal neuronal differentiation. The transcriptional programs regulated by RP58 during this process are not known. Here, we report for the first time that in embryonic mouse neocortical neurons a complex set of genes normally expressed in other cell types, such as those from mesoderm derivatives, must be actively repressed in vivo and that RP58 is a critical regulator of these repressed transcriptional programs. Importantly, the GSEA analyses of these transcriptional programs indicate that repressed genes include distinct sets of genes significantly associated with glioma progression and/or pluripotency. We also demonstrate that reintroducing RP58 in glioma stem cells not only leads to aspects of neuronal differentiation but also to loss of stem cell characteristics, including loss of stem cell markers and decrease in stem cell self-renewal capacities. Thus, RP58 acts as an in vivo master guardian of the neuronal identity transcriptome and its function may be required to prevent brain disease development including glioma progression.