Differentiation-dependent thymocyte migration in the thymus may be important for T lymphopoiesis and might be regulated by thymic chemoattractants. We examined modulation of chemotactic responsiveness of thymocyte subsets during their early to late stages of development in response to 2 thymus-expressed chemokines, SDF-1 and CKβ-11/MIP-3β/ELC. SDF-1 shows chemotactic preference for immature thymocytes (subsets of triple negative thymocytes and double positive [DP] subset) over mature single positive (SP) thymocytes. CKβ-11/MIP-3β/ELC shows low chemotactic activity on the immature thymocytes, but it strongly attracts mature SP thymocytes, effects opposite to that of SDF-1. SDF-1–dependent chemoattraction of immature thymocytes is not significantly desensitized by a negative concentration gradient of CKβ-11/MIP-3β/ELC, and chemoattraction of mature SP thymocytes to CKβ-11/MIP-3β/ELC is not antagonized by SDF-1, demonstrating that these two chemokines have different chemoattractant preferences for thymocyte subsets and would probably not inhibit each other's chemotaxis in the event of microenvironmental coexpression. The chemotactic responsiveness of thymocytes and mature T cells to the 2 chemokines is respectively enhanced after selection process and migration to the spleen. These studies demonstrate the presence of thymocyte chemoattractants with differential chemotactic preference for thymocytes, a possible mechanism for thymocyte migration in the thymus.