Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 microm2 spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 microm2 failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-microm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 microm2. Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-microm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 microm2), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis-growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.