The Mrj co-chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj−/− embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin-walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down-regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj−/− neuroepithelial cells. Furthermore, Mrj−/− neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self-renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi-1, a self-renewal protein. Furthermore, unlike in Mrj−/− placentas, intermediate filament-containing aggregates do not accumulate in Mrj−/− neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self-renewal. Developmental Dynamics 238:2564–2574, 2009. © 2009 Wiley-Liss, Inc.