The transcription factor family of nuclear factor I (NFI) proteins is encoded by four closely related genes: Nfia, Nfib, Nfic, and Nfix. A potential role for NFI proteins in regulating developmental processes has been implicated by their specific expression pattern during embryonic development and by analysis of NFI-deficient mice. It was shown that loss of NFIA results in hydrocephalus and agenesis of the corpus callosum and that NFIB deficiency leads to neurological defects and to severe lung hypoplasia, whereas Nfic knockout mice exhibit specific tooth defects. Here we report the knockout analysis of the fourth and last member of this gene family, Nfix. Loss of NFIX is postnatally lethal and leads to hydrocephalus and to a partial agenesis of the corpus callosum. Furthermore, NFIX-deficient mice develop a deformation of the spine, which is due to a delay in ossification of vertebral bodies and a progressive degeneration of intervertebral disks. Impaired endochondral ossification and decreased mineralization were also observed in femoral sections of Nfix-/- mice. Consistent with the defects in bone ossification we could show that the expression level of tetranectin, a plasminogen-binding protein involved in mineralization, is specifically downregulated in bones of NFIX-deficient mice.