Mast cells are widely distributed throughout the body, predominantly near blood vessels and nerves, and express effector functions in allergic reactions, inflammatory diseases, and host defense. The activation of mast cells results in secretion of the preformed chemical mediators in their granules by a regulated process of exocytosis and leads to synthesis and secretion of lipid mediators and cytokines. Their soluble factors contribute to allergic inflammation. Mast cells are associated with hypersensitivity reactions, not only in the classical immunoglobulin E (IgE)-dependent mechanism but also in an IgE-independent manner. In particular, investigations of potential anatomical and functional interactions between mast cells and the nervous system have recently attracted great interest. To understand these molecular mechanisms in mast cell activation, the ability to visualize, track, and quantify molecules and events in living mast cells is an essential and powerful tool. Recent dramatic advances in imaging technology and labeling techniques have enabled us to carry out these tasks with high spatiotemporal resolution using confocal laser scanning microscopes, green fluorescent protein and its derivatives, and image analysis systems. Here we review our investigations of the dynamic processes of intracellular signaling molecules, cellular structure, and interactions with neurons in mast cells to provide basic and valuable information for allergy and clinical immunology using these new imaging methods.