Trafficking of lymphocytes through lymphatics and secondary lymphoid organs is crucial for immunity. While the process of T lymphocyte migration across high endothelial venules (HEV) into lymph nodes (LN) is well characterized, relatively little is known about the mechanisms that regulate migration of lymphocytes from tissues into afferent lymphatics. Several groups have attempted to develop in vitro methods to investigate lymphatic migration. Culture of freshly isolated lymphatic endothelial cells has proven very difficult, and ultimately no cell line produced has been shown to retain a lymphatic lineage after multiple rounds of passage. The C3H/HeJ mouse endothelial line SVEC4-10 is an SV-40 transformant of endothelial cells isolated from the axillary LN, and has been variously reported as lymphatic, HEV, or blood microvascular endothelium. We have characterized this cell line via flow cytometry, RT-PCR, and gene array analysis, and have determined that it represents a lymphatic endothelial cell lineage. We have also shown that migration of T cells across this cell line in vitro recapitulates in vivo migration of T cells, across lymphatic endothelial cells such that T cells migrate only in a basal (abluminal) to apical (luminal) direction. This protocol outlines the methods for manipulating these cells and performing transendothelial migration.