Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation. Wild type (WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6-72 hours. Ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared to WT and Notch3-/-. Significantly greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT mice after ozone. Expression of whole lung Tnf was significantly increased after ozone in all genotypes, and was significantly greater in Notch3-/- mice compared to WT. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member. These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.