Adaptation of a cell behavior to the environment is possible due to the biochemical and physical information that is transmitted through molecular receptor present at the cell surface. Regulation of receptor distribution and trafficking is thus a key feature to allow cells to properly respond to extracellular signals. Many of the molecular mechanisms that support receptor trafficking occurs at a submicrometric scale and are highly dynamic. Because of its exceptional resolution and its piconewton sensitivity, atomic force microscope (AFM) is a powerful tool to study the trafficking of individual receptors in living cells under near-physiological conditions. In this review, we first describe the general principles of the AFM that allow the detection of single ligand-receptor interaction. We then turn to early studies that demonstrated the ability of AFM to detect individual receptors and map their distribution on the surface of living cell. Finally, we discuss how AFM in combination with optical imaging tools allow the simultaneous investigation of cellular biophysical properties and receptor-trafficking dynamics at the nanometer scale.