Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein–coupled receptors, which are linked to the heterotrimeric G proteins G_q/G₁₁, G₁₂/G₁₃, and G_i. The role of downstream signaling pathways activated by anaphylactic mediators in defined organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of G_q/G₁₁- and G₁₂/G₁₃-mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that G_q/G₁₁-mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various inflammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial Gα_q/Gα₁₁ deficiency. Mice with endothelium-specific Gα_q/Gα₁₁ deficiency, but not with Gα₁₂/Gα₁₃ deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial G_q/G₁₁-mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions.