The murine winged helix gene Fkh5 is specifically expressed in the developing central nervous system (CNS). Early embryonic Fkh5 expression is restricted to the mammiliary body region of the caudal hypothalamus, midbrain, hindbrain and spinal cord. Postnatally, signals persist in specific nuclei of the mammillary body and in the midbrain. We generated Fkh5 deficient mice by homologous recombination to assess its in vivo function. At birth, Fkh5-deficient mice are viable and indistinguishable from wild-type and Fkh5 heterozygous littermates. However, about one third die within the first two days and another fifth before weaning. Surviving Fkh5-deficient mice become growth retarded within the first week and remain smaller throughout their whole life span. Fkh5-deficient females on 129Sv x C57BL/6 genetic background are fertile, but do not nurture their pups. More detailed analysis of Fkh5-deficient brains reveals distinct alterations in the CNS. In the midbrain, mutant mice exhibit reduced inferior colliculi and an overgrown anterior cerebellum. Furthermore, the hypothalamic mammillary body of Fkh5-deficient brains lacks the medial mammillary nucleus. These results suggest that Fkh5 plays a major role during CNS development.