Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS−/− mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4+ Foxp3+ regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS−/− mice the number of CD4+ CD25+ Foxp3+ Tregs did not increase after respiratory Ag application, and ICOS−/− Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS−/− Tregs did not convey protection when transferred to “asthmatic” recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.