Histone H2AX plays a crucial role in molecular and cellular responses to DNA damage and in the maintenance of genome stability. It is downstream of ataxia telangiectasia mutated (ATM) damage signaling pathway and there is an emerging role of the transcription factor FoxO3a, a regulator of a variety of other pathways, in activating this signaling. We asked whether H2AX may feedback to FoxO3a to affect respective FoxO3a-dependent pathways. We used a genetically matched pair of mouse embryonic fibroblast H2AX(+/+) and H2AX(-/-) cell lines to carry out comprehensive time-course and dose-response experiments and to show that the expression of several FoxO3a-regulated genes was altered in H2AX(-/-) compared to H2AX(+/+) cells at both basal and irradiated conditions. Hspa1b and Gadd45a were down-regulated four- to five-fold and Ddit3, Cdkn1a and Sod2 were up-regulated 2-3-fold in H2AX(-/-) cells. Using the luciferase reporter assay, we directly demonstrated that transcriptional activity of FoxoO3a was reduced in H2AX(-/-) cells. FoxO3a localization within the nuclear phospho-ATM (Ser1981) foci in irradiated cells was affected by the H2AX status, as well as its posttranslational modification (phospho-Thr32). These differences were associated with genomic instability and radiosensitivity in H2AX(-/-) cells. Finally, knockdown of H2AX in H2AX(+/+) cells resulted in FoxO3a-dependent gene expression patterns and increased radiosensitivity that partially mimicked those found in H2AX(-/-) cells. Taken together, our data suggest a role for FoxO3a in the maintenance of genome integrity in response to DNA damage that is mediated by H2AX via yet unknown mechanisms.