The polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed detectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16(Ink4a) and p19(Arf) in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.