The body’s defense against pathogens relies on global surveillance by immune cells. Leukocyte trafficking is a dynamic complex phenomenon depending on a network of numerous cell subtypes in relation with their anatomical environment. Leucocytes travel back and forth between the tissues and lymphoid organs via lymphatic and blood vessels, within which they are carried passively by the flow and actively by their own capacity to self-propel through adhesive migration on vessel walls and within tissues. The regulation of adhesion, migration and guidance of immune cells is critical for many pathophysiological situations including immune deficiencies, tumor cell invasion, atherosclerosis or multiple sclerosis. However the mechanisms underlying leukocyte trafficking remain poorly understood. In vivo, observations of these dynamic cell processes using for instance microscopy imaging are difficult to obtain. Moreover, the interpretation of such data is extremely complex due to the many uncontrolled stimuli that govern immune cell trafficking. We therefore develop in vitro approaches to analyze leukocyte migration and guidance mechanisms in controlled microenvironment and at the single cell level. We use advanced techniques such as microfluidics, optical protein printing, and single cell micromanipulation to investigate the deformation of leucocytes in microvasculature and tissue matrix, monitor cell arrest and migration on blood vessel wall and probe cell guidance and response to mechanical and chemical stimuli.