CCL21 (SLC/6Ckine) is constitutively expressed by secondary lymphoid tissue and attracts CCR7-expressing mature dendritic cells and naive T cells. Recent studies demonstrated that intra-tumoral delivery of CCL21 induces tumor regression in a T cell dependent manner. CCL21 is known to mediate T cell trafficking but little is known about its function as a costimulatory molecule. Herein, we demonstrate that CCL21 costimulates expansion of CD4+ and CD8+ T cells and induces Th1 polarization. These effects were specific for naive T cells, and we show that CD4+CD25+ regulatory T cells were hyporesponsive to CCL21 induced migration, and unresponsive to CCL21 costimulation. These unique functions of CCL21 to both attract naive T cells as well as costimulate their proliferation and differentiation, suggests that CCL21 is a pivotal molecule for priming T cell responses and has therapeutic implications for local delivery of CCL21. The coordinated effects of CCL21 on T cell migration and activation may also represent a more comprehensive paradigm for the activity of other chemokines as well.