OBJECTIVE Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme with immunoregulatory properties in cancer. By focusing on multiple myeloma cells and its microenvironment as potential sources of IDO, we aimed to delineate its influence on myeloma cell growth and survival and examine effector mechanisms. METHODS IDO expression was assessed in myeloma cells and in a coculture system with mesenchymal stromal cells (MSCs), including prior cytokine priming to induce IDO in MSCs. IDO expression was correlated with induction of apoptosis in myeloma cells and coupled with tryptophan depletion as well as rescue using IDO inhibitors. RESULTS We report low levels of expression of IDO in myeloma cell lines (MMCLs) and primary myeloma cells (MMCs), despite priming with interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or hepatocyte growth factor (HGF). In MSCs, however, IDO could be functionally induced by IFN-γ, mediating apoptosis in myeloma cells following coculture. Addition of IDO-specific inhibitors, as well as addition of tryptophan, was shown to abrogate these effects. CONCLUSIONS IDO is expressed in primary MMCs to a low degree and is unlikely to play a direct major role in vivo in dampening antitumor immunity. However, cytokine stimulation of MSCs specifically induced IDO, which mediated a marked sensitivity of proximal myeloma cells to tryptophan depletion in the microenvironment, suggesting that selective measures to regulate its availability could be a useful strategy to achieve myeloma growth inhibition and apoptosis.