The presence of Langhans giant cells (LGCs) is one of the signatures of systemic granulomatous disorders such as tuberculosis and sarcoidosis. However, the pathophysiological mechanism leading to LGC formation, especially the contribution of the T cells abundantly found in granulomas, has not been fully elucidated. To examine the role of T cells in LGC formation, a new in vitro method for the induction of LGCs was developed by co-culturing human monocytes with autologous T cells in the presence of concanavalin A (ConA). This system required close contact between monocytes and T cells, and CD4+ T cells were more potent than CD8+ T cells in inducing LGC formation. Antibody inhibition revealed that a CD40-CD40 ligand (CD40L) interaction and IFN-γ were essential for LGC formation, and the combination of exogenous soluble CD40L (sCD40L) and IFN-γ efficiently replaced the role of T cells. Dendritic cell-specific transmembrane protein (DC-STAMP), a known fusion-related molecule in monocytes, was up-regulated during LGC formation. Moreover, knock-down of DC-STAMP by siRNA inhibited LGC formation, revealing that DC-STAMP was directly involved in LGC formation. Taken together, these results demonstrate that T cells played a pivotal role in a new in vitro LGC formation system, in which DC-STAMP was involved, and occurred via a molecular mechanism that involved CD40-CD40L interaction and IFN-γ secretion.