The immunomodulatory properties of mesenchymal stem/stromal cells (MSCs) make them an attractive therapeutic tool to treat chronic inflammatory diseases, such as rheumatoid arthritis or Crohn's disease. These indications are characterized by a chronic activation of immune cells that perpetuates the disease. In vitro, when adipose mesenchymal stem cells (ASCs) are cultured with T lymphocytes at the time of stimulation, their proliferation is inhibited. However, these experimental settings do not necessarily fit with what ASCs will face in inflammatory conditions in vivo, where ASCs will likely encounter and interact with already activated immune cells which might affect their immunomodulatory capacity. In most in vitro studies, MSCs have been cultured with peripheral blood mononuclear cells at the time of lymphocyte stimulation and information about the interaction between MSCs and prestimulated lymphocytes in vitro is scarce. Therefore, a better understanding of the capacity of MSCs to modulate the responses of preactivated immune cells is needed. In this study we focused on the effects of ASCs on prestimulated lymphocytes and systematically investigated the potential mechanisms involved. We report that prestimulation of T lymphocytes 48 h before the coculture with ASCs impairs the capacity of ASCs to inhibit proliferation. Preactivation of ASCs with interferon γ or the toll-like receptor ligand Poly I:C, but not other stimuli tested, enhanced the ability to inhibit the proliferation of 48 h-stimulated T lymphocytes. The inhibitory effect of ASCs was shown to be time dependent and mediated through the actual magnitude of tryptophan degradation by indoleamine 2,3-dioxygenase.