Cancer immunotherapy activates the immune systems to treat the tumor and has changed the paradigm of existing anti-cancer drugs that have side effects and resistance. However, the therapy has been reported to show different efficacy for each patient, and various approaches and understandings of the tumor microenvironment are required, which is one of the major immunosuppression mechanisms. This thesis describes the development of microfluidic systems capable of three-dimensional co-culture of multiple cell types (e.g. tumor, endothelial, and immune cells) in microchannels and precise control of microenvironmental variables. The microfluidic systems present in vitro applications of reconstitution of immune cell apoptosis by tumor vasculatures, recapitulation of drug resistance in tumor microenvironment, and acoustofluidic-based dynamic culture of immune cells.