Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19+ DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19+ DC, IFN typeI (IFN-) is the obligate inducer of IDO. We now report that IFN- production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity. Genetic and pharmacologic ablation of IDO completely abrogated IFN- production by CD19+ DC after B7 ligation. In contrast, IDO ablation did not block IFN- production by CD19+ DC after TLR9 ligation. IDO-mediated control of IFN- production depended on tryptophan depletion as adding excess tryptophan also blocked IFN- expression after B7 ligation. Consistent with this, DC from mice deficient in general control of non-derepressible-2 (GCN2)-kinase, a component of the cellular stress response to amino acid withdrawal, did not produce IFN- following B7 ligation, but produced IFN- after TLR9 ligation. Thus, B7 and TLR9 ligands stimulate IFN- expression in CD19+ DC via distinct signaling pathways. In the case of B7 ligation, IDO activates cell-autonomous signals essential for IFN- production, most likely by activating the GCN2-kinase-dependent stress response. See accompanying commentary: http://dx.doi.org/10.1002/eji.20073737184 .