Defects of the intracellular enzyme 3' repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi-Goutières syndrome and are associated with systemic lupus erythematosus. Trex1(-/-) mice develop type I IFN-driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP-AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA.