T-cell motility is critical for leukocyte trafficking both in normal host defense and in pathologic conditions including chronic inflammatory disease. Despite progress in understanding the mechanisms of T-cell polarity and motility, we have limited understanding of the mechanisms that contribute to antigen-induced T cell arrest. Here, we describe methods to analyze leukocyte function antigen-1-mediated T-cell motility and T-cell receptor-induced stop signals using in vitro assays on two-dimensional surfaces. Specifically, methods for live time-lapse imaging of T cell random migration and arrest on ICAM-1-coated surfaces are described. Additionally, we detail methods for live imaging of T-cell motility within 3D substrates to analyze T cell-antigen-presenting cell (APC) interactions and APC-mediated stop signals.