Simple SummaryAtomic force microscopy (AFM) is a powerful technique that has been pivotal to cancer research, but it has only recently been used to study tumor pathology at the tissue scale. In this Review, we highlight studies that have used AFM to characterize the mechanical properties of various cancer tissues and discuss the application of this methodology in the clinic.AbstractAtomic force microscopy (AFM) is a popular tool for evaluating the mechanical properties of biological materials (cells and tissues) at high resolution. This technique has become particularly attractive to cancer researchers seeking to bridge the gap between mechanobiology and cancer initiation, progression, and treatment resistance. The majority of AFM studies thus far have been extensively focused on the nanomechanical characterization of cells. However, these approaches fail to capture the complex and heterogeneous nature of a tumor and its host organ. Over the past decade, efforts have been made to characterize the mechanical properties of tumors and tumor-bearing tissues using AFM. This has led to novel insights regarding cancer mechanopathology at the tissue scale. In this Review, we first explain the principles of AFM nanoindentation for the general study of tissue mechanics. We next discuss key considerations when using this technique and preparing tissue samples for analysis. We then examine AFM application in characterizing the mechanical properties of cancer tissues. Finally, we provide an outlook on AFM in the field of cancer mechanobiology and its application in the clinic.