The danger theory of immune tolerance asserts that environmental factors hold primacy over lymphocyte autoreactivity in initiating autoimmune disease. We sought to test this contention using the Aire-deficient mouse model of the human disease, autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy, a multiorgan autoimmune disorder rooted in a lesion in thymic tolerance. Compound screens stimulating a broad range of innate immune system pathways failed to show any modulation of disease characteristics inAire−/−mice on either the C57BL/6 or NOD genetic backgrounds. Furthermore, deficiency in the Toll-like receptor adaptor Myd88 increased the lifespan of NOD.aire−/−mice but did not prevent the initiation of autoimmunity. Finally, germ-free NOD.aire−/−mice exhibited autoimmunity in all organs normally targeted in this model, indicating that microbial conditioning is not required for activation of autoreactive T cells relevant to this disease. Together, these data suggest that the stochastic genesis of dangerous T cell clones can initiate autoimmune disease without the need for environmental stimulation, underlining the importance of Aire-dependent thymic deletion.