The thymic architecture is normally compartmentalized into a central medulla surrounded by a peripheral cortical region. We investigated how compartmentalization of the thymic stroma is regulated using T cell receptor (TCR)‐transgenic mouse models. Our studies show that the signals generated by TCR/peptide/major histocompatibility complex interactions regulate thymic stromal cell compartmentalization. In TCR‐transgenic mice, normal stromal cell compartmentalization occurs when the transgenic TCR is expressed on a background that does not result in skewing toward either positive or negative selection. In models representing strong positive selection, the thymic stromal elements do not fully organize into a central medulla. Instead, small medullary foci are dispersed throughout the thymus with some regions residing directly under the capsule. The highest degree of disorganization in medullary epithelial regions is observed in TCR‐transgenic mice that exhibit negative selection. Although the medullary foci lack central organization, the expression in these regions of CD80, CD86 and CD40, as well as the clustering of dendritic cells, is similar to that observed in medullae of wild‐type mice. Thus, the organization of the medulla appears to occur in two stages: (1) small medullary epithelial regions that are dispersed in fetal thymi expand and associate with antigen‐presenting cells, and (2) the expanded medullary foci organize into a central medullary compartment. Our data suggest a model in which this second stage of stromal cell organization is increasingly inhibited as the normal balance of TCR‐mediated signals is skewed by higher‐avidity interactions between thymocytes and antigen‐presenting cells.