Oxidative stress in allergic asthma may result from oxidase activity or pro-inflammatory molecules in pollens. Signaling via TLR4 and its adaptor TRIF has been implicated in reactive oxygen species (ROS)-mediated acute lung injury and in T helper 2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine (NAC) or dimethylthiourea (DMTU) prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. NAC inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas DMTU was inhibitory even when administered with the sensitization alone. Heat-inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4- and TRIF-independent, however, TLR4 deficiency decreased, while TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation which is restrained by the TRIF-dependent pathway.