Studies on thymopoiesis are critical to the understanding of T-cell homeostasis as well as the host response to T-cell depletion. Various in vitro culture systems have been used in the study of thymocyte development; however it is unclear if current co-culture methods have been fully optimized. In this study in vitro suspension cultures have been re-evaluated and the optimal storage conditions for thymocytes have been established by evaluating various methods of storing/isolating thymic tissue and isolated thymocytes as well as the source of thymic epithelial cells (TEC). It was determined that thymocytes must be freshly isolated from whole thymic tissue and ideally stored at 4 degrees C prior to co-culture. Co-culture with either autologous or allogeneic TEC results in similar thymocyte subset distribution as well as interleukin-7 receptor-alpha (CD127) expression on these subsets. To evaluate the influence of the source of TEC on one aspect of thymocyte function the effect of IL-7 stimulation on the expression of CD127 was evaluated. IL-7 stimulation resulted in a downregulation of the expression of CD127 on all thymic subsets similar to that observed in circulating CD8+ T-cells. The effect of this was the same whether TEC were autologous or allogeneic. Optimizing culture techniques and facilitating the study of individual thymocyte subsets will lead to a better understanding of thymic function and development. It could also lead to therapeutic approaches that enhance immune recovery after T-cell depletion in HIV infection, bone marrow transplantation or following chemotherapy.