The immune system is composed of heterogeneous populations of immune cells that regulate physiological processes and protect organisms against diseases. Single cell technologies have been used to assess immune cell responses at the single cell level, which are crucial for identifying the causes of diseases and elucidating underlying biological mechanisms to facilitate medical therapy. In the present review we first discuss the most recent advances in the development of single cell technologies to investigate cell signaling, cell-cell interactions and cell migration. Each technology's advantages and limitations and its applications in immunology are subsequently reviewed. The latest progress toward commercialization, the remaining challenges and future perspectives for single cell technologies in immunology are also briefly discussed.