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Tunneling nanotubes (TNT) are the latest addition to the array of strategies used for intercellular signaling. TNTs are continuous conduits of the plasma membrane that allow direct physical connection of plasma membranes and cytosol among remote cells. They are important for intercellular communication by mediating exchange of cellular components as well as signal transduction molecules. Despite ample evidence suggesting the pathophysiological importance of TNTs, virtually nothing is known about the molecular basis for their formation. With the lack of specific TNT markers, their study has relied solely on morphological analyses, and the precise identity of TNT and TNTlike structures have been difficult to define. We have now shown that M-Sec is a TNT marker and a central factor for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton is involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels.