Infection of both lymphoid and stromal components of the thymus by human immunodeficiency virus type 1 (HIV-1) suggests that impairment of lymphocyte differentiation from early T cells progenitors in the thymus may contribute to the HIV-induced T cell depletion. Cross-talk between immature thymocyte and thymic epithelium through cell-to-cell adhesion mediated by fibronectin/receptor interaction plays a central role in driving T cell development. HIV-1 tat protein, like fibronectin, contains an RGD sequence involved in the interaction with fibronectin receptor. We demonstrated that gene transfer-mediated tat expression in thymic stroma is able to influence the in vitro maturation of T cell progenitors as tat-expressing epithelial cells have a decreased ability to drive the generation of CD4+8+ thymocytes from CD4-8- precursors. Furthermore, tat-expressing cells produce more fibronectin and display upregulation of VLA-5 cell surface receptor levels compared to control cells, while alpha v expression was unchanged. Cellular distribution of fibronectin is also influenced by tat. Fibronectin is distributed in the whole cell surface and along cell processes in control cells whereas it is mainly concentrated in the intracytoplasmic area in tat-expressing cells. Therefore, expression of tat in thymic epithelial cells impairs thymocyte maturation and modulates fibronectin expression: this suggests a crucial role of this viral protein in regulating the T lymphocyte differentiation program through modulation of intrathymic lympho-stromal interactions.