Integrin-extracellular matrix (ECM) interactions in two-dimensional (2D) culture systems are widely studied (Goldstein and DiMilla, 2002. J Biomed. Mater. Res. 59, 665–675; Koo et al., 2002. J. Cell Sci. 115, 1423–1433). Less understood is the role of the ECM in promoting intercellular cohesion in three-dimensional (3D) environments. We have demonstrated that the α5β1-integrin mediates strong intercellular cohesion of 3D cellular aggregates (Robinson et al., 2003. J. Cell Sci. 116, 377–386). To further investigate the mechanism of α5β1-mediated cohesivity, we used a series of chimeric α5β1-integrin–expressing cells cultured as multilayer cellular aggregates. In these cell lines, the α5 subunit cytoplasmic domain distal to the GFFKR sequence was truncated, replaced with that of the integrin α4, the integrin α2, or maintained intact. Using these cells, α5β1-integrin–mediated cell aggregation, compaction and cohesion were determined and correlated with FN matrix assembly. The data presented demonstrate that cells cultured in the absence of external mechanical support can assemble a FN matrix that promotes integrin-mediated aggregate compaction and cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction, resulting in cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.