T-cell development is under the tight control of thymic microenvironments. Conversely, the integrity of thymic microenvironments depends on the physical presence of developing thymocytes, a phenomenon designated as ‘thymic crosstalk’. We now show, using three types of immunodeficient mice, i.e. CD3ε transgenic mice, RAG^null mice and RAG^null-bone-marrow-transplanted CD3ε transgenic mice, that the control point in lymphoid development where triple negative (CD3⁻,CD4⁻,CD8⁻) thymocytes progress from CD44⁺CD25⁻ towards CD44⁻ CD25⁺, influences the development of epithelial cells, critically inducing the extra, third dimension in the organization of the epithelial cells in the cortex. This tertiary configuration of the thymic epithelium is a typical feature for the thymus, enabling lymphostromal interaction during T-cell development. Crosstalk signals at this control point also induce the formation of thymic nurse cells. Moreover, our data indicate that establishment of a thymic cortex is a prerequisite for the development of the thymic medulla. Thus, differentiating thymocytes regulate the morphogenesis of thymic microenvironments in a stepwise fashion.