Dendritic cell (DC) licensing in cross-priming requires physical interaction of several rare immune cells, i.e. cytotoxic T cells (CTL), and cross-presenting DCs. Here we describe a novel in vitro method of analyzing chemokine effects on complex recruitment events in a multi-cellular system. To study CTL recruitment towards CCL17-producing DCs, we established a co-culture system of murine splenic DCs with polyclonal splenic CTL from donor mice, which enables visualization of cell motility and cell-cell interactions at a high resolution over a period of several hours by use of multi-color live cell imaging. Differential cell labeling with plasma membrane-permeable cell tracker dyes was employed for the specific detection of each cell population. Mixed cell populations were placed on fibronectin-coated channel slides following recording of time-lapse video series with a fully automated inverted microscope. Motile CTL were individually tracked using ImageJ software with subsequent calculation of directionality and velocity of migrating CTL before physical contact to DC. The duration of cell-cell interactions was determined as well (contact duration time). This procedure enables simultaneous analyses of cell velocity, migratory directionality and cell contact duration times in e.g. a three-partite system comprising DC, as well as wt and chemokine receptor null cytotoxic T cells.