During thymus development, prothymocytes home to the thymus where they migrate as maturing thymocytes from the cortex to the medulla. Chemotaxis assays show that developing T cells of newborn mice respond to certain chemokines depending on their differentiation state. In situ expression analyses indicate that the same chemokines are expressed in distinct microenvironments within the thymic stroma. Expression of chemokines is regulated temporally during embryogenesis; in the alymphoid early thymic anlage, only TECK, SDF-1 and SLC but not ELC, MDC or TARC are expressed. Fetal blood prothymocytes destined to colonize the thymus respond to the embryonic chemokines TECK and SDF-1 in chemotaxis assays with high efficacy. The in vivo significance of this finding is demonstrated by studies in the nude mouse where the thymic anlage lacks TECK and SDF-1 expression and prothymocytes home to the parathyroid anlage rather than to the thymic anlage. Developing thymocytes respond to chemokines expressed in distinct microenvironments within the thymic stroma in a way that correlates well with the previously observed migration pattern from cortex to medulla. The complexity of these chemokine-defined microenvironments increases as the thymic anlage develops to a mature thymus.