AbstractPolynucleotide kinase–phosphatase (PNKP) is a DNA repair factor possessing both 5′‐kinase and 3′‐phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame‐shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non‐homologous end‐joining, pathways required for repair of both DNA single‐ and double‐strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease.