The in vitro culture system outlined in this chapter allows for standardized protocols to examine canonical and adaptive natural killer (NK) cell responses while interacting with immune suppressor cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). These interactions pathologically occur during tumorigenesis and tumor progression. Strategies to study the effects of ex vivo purified immune suppressor cells from cancer patients on the function of NK cell antitumor activity will help to understand suppressive mechanisms to improve immunotherapy. Immune checkpoint inhibitors have recently demonstrated tremendous clinical responses in patients with diverse types of cancers. However, their effect on NK cell function is not very well studied. Here, we have adapted a coculture system that previously has been utilized to study regulatory T cells. This approach can further be utilized to study the effects of immune checkpoint inhibitors in vitro and ex vivo. We focus on the differences between canonical NK cells and the newly identified subset of NK cells termed “adaptive NK cells.” These cells are induced by cytomegalovirus (CMV) in CMV-seropositive individuals.