A cell in vivo is part of a large, networked community. An individual cell’s fate is strongly influenced by its interactions with neighbouring cells. While this interaction has been recognized as critical in determining cell behaviour, its complexity and heterogeneity has thus far defied characterization by currently available techniques. Herein, we present a single-cell level co-culture platform for studies of dynamic cellular interactions, which is capable of maintaining and tracking single-cell pair interactions to simplify the complexity of intercellular communication. In this platform, heterotypic pairing on a single-cell level is achieved through sequential cell trapping and dynamic variation of fluidic resistance. Individual culture chambers provide trapped cells enough space to migrate and proliferate through multiple generations. Furthermore, the semi-isolated chambers, combined with continuously refreshed medium supplement, allow a stable communication environment around the cells. To demonstrate the platform capability, we cultured and tracked stem cell-fibroblast pairs for several generations. The subsequent effects of cell–cell interactions were then easily observed, due to the addressability of each isolated chamber, and quantitatively characterized. Specifically, we found that paired cells’ migration patterns were dependent on their initial distance from each other, and that heterotypic pairing led to distinct proliferation patterns from homotypic, single-cell culture. This study demonstrates the platform utility in providing a detailed and quantitative understanding of the complexity of cellular communication and its effects on cell behaviour in a variety of biological systems.