Human T lymphocytes expressing the Vγ9Vδ2 TCR recognize non-peptidic Ags, referred to as phosphoantigens, produced by microbial pathogens and by human tumor cells. Here we show that γδ T cells establish a mature immunological synapse (IS) with the myelomonocytic THP-1 tumoral cell line. This synapse is characterized by an enrichment for phosphotyrosine, CD2, and γδ TCR together with the exclusion of CD45. The CD94 and NKG2D receptors are also recruited to the signaling area, while the C-lectin-like activation marker CD69 segregates out of the synapse. γδ T cell conjugation to THP-1 increases upon stimulation by soluble phosphoantigen, is paralleled by the metabolic activation of γδ T cells and leads to cytokine production. Molecular segregation of the above molecules also occurs at the γδ T cell/THP-1 interface in the absence of exogenously added phosphoantigen, although it does not result in intracellular signaling and cytokine production under these conditions. Hence the molecular interactions at the γδ T cell-THP-1 target cell interface are sufficient to induce the formation of an IS, but cytokine production requires the full engagement of γδ TCR by a strong agonist. Thus in γδ T cells, formation of the IS is uncoupled from its functional outcome.