Runt-related transcription factor 1 (RUNX1), a transcription factor expressed in multiple organs, plays important roles in embryonic development and hematopoiesis. Although RUNX1 is highly expressed in pulmonary tissues, its roles in lung function and homeostasis are unknown. We sought to assess the role of RUNX1 in lung development and inflammation after LPS challenge. Expression of RUNX1 was assessed in the developing and postnatal lung. RUNX1 was conditionally deleted in pulmonary epithelial cells. Pulmonary maturation was evaluated in the developing and postnatal lung, and lung inflammation was investigated in adult mice after LPS challenge. Interactions between RUNX1 and inflammatory signaling via NF-kappa B-IkB kinase beta were assessed in vitro. RUNX1 was expressed in both mesenchymal and epithelial compartments of the developing and postnatal lung. The RUNX1 gene was efficiently deleted from respiratory epithelial cells producing Runx1(Delta/Delta) mice. Although lung maturation was delayed, Runx1(Delta/Delta) mice survived postnatally and subsequent growth and maturation of the lung proceeded normally. Increased respiratory distress, inflammation, and proinflammatory cytokines were observed in the Runx1-deleted mice after pulmonary LPS exposure. RUNX1 deletion was associated with the activation of NF-kappa B in respiratory epithelial cells. RUNX1 was required for the suppression of NF-kappa B signaling pathway via inhibition of IkB kinase beta in in vitro studies. RUNX1 plays a critical role in the lung inflammation after LPS-induced injury.