We have investigated the role of specific components of the thymic stroma during development of CD4(-)8(-) T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25(+) 44(+) precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25(+) 44(-) precursors, develop to the CD4(+) 8(+) stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25(+) 44(+) precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4(+) 8(+) cells are generated from CD25(+) 44(+) precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.