The mechanisms underlying leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.