Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common cellular pathway. To further understand the in vivo role of one of these human genes (FANCG), we generated a targeted disruption of murine Fancg and bred mice homozygous for the targeted allele. Similar to the phenotype of the previously described Fancc^−/− andFanca^−/− mice, theFancg^−/− mice had normal viability and no gross developmental abnormalities. Primary splenic lymphocytes, bone marrow progenitor cells, and murine embryo fibroblasts from theFancg^−/− mice demonstrated spontaneous chromosome breakage and increased sensitivity to mitomycin C and, to a lesser extent, ionizing radiation. Fancg^−/−lymphocytes had a defect in the FA pathway, based on their failure to activate the monoubiquitination of the downstream Fancd2protein in response to IR. Finally,Fancg^−/− mice had decreased fertility and abnormal gonadal histology. In conclusion, disruption of theFancg gene confirms the role of Fancg in the FA pathway. The Fancg^−/− mouse may be useful as an animal model for future gene therapy and cancer susceptibility studies.