Phosphorylated nonpeptide compounds have recently been identified as potent mycobacteria-derived ligands for human Vy9N62-expressing y6 T cells. Crude mycobacterial extracts also contain protein antigens which stimulate CD4 ap T cells to produce growth factors that are used by y6 T cells for clonal expansion. We have investigated the dynamics in vitro of expansion of CD4 T cells and Vy9 cells in cultures of peripheral blood mononuclear cells stimulated with synthetic isopentenyl pyrophosphate (IPP) in the absence or presence of additional stimuli. The results indicated that following stimulation with IPP, y6 T cells express CD25 and CD69 antigens, but fail to proliferate unless growth factors are provided exogenously or endogenously through activation of CD4 T cells by additional stimuli such as tetanus toxoid, alloantigen, or superantigens. Furthermore, the presence of antigen presenting cells are required for expansion of y6 T cells. In response to IPP stimulation, purified CD4 T cells neither express CD25 or CD69, nor do they proliferate even in the presence of exogenous IL-2. Apart from IL-2, IL-15 and, less efficiently, IL-4, IL-7, and IL-12 can contribute to cellular expansion of IPP-reactive Vy9 cells. Together, the results demonstrate that peripheral blood y6 T cells proliferate in response to IPP only if CD4 T cells are simultaneously activated by an additional stimulus. This mechanism provides a tight control of the reactivity of y6 T cells towards phosphorylated nonpeptide antigens.