Naive T lymphocytes traffic through the organism in their search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on the chemokine CCL21, while exit into efferent lymphatics relies on the sphingolipid S1P. Surprisingly, while both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist in vitro approach to study the real-time, single-cell response of naive T lymphocytes to CCL21 and S1P-rich serum. Using high-throughput microfluidic and optical micropatterning ad hoc tools, we show that CCL21 triggers long-range chemotaxis whereas S1P-rich serum does not. Instead, S1P-rich serum triggers a transient polarization that may represent a brief transmigration step through exit portals. Our data thus validate naive T lymphocyte chemotaxis towards CCL21 but not S1P, which complements in vivo observations and is of interest for a better tailoring of immunosuppressive drugs.