The lymphotoxin LTα1β2 supports the development and maintenance of several aspects of spleen structure, but its significance for marginal sinus (MS) vascular organization is unclear. We showed here that, in early postnatal lymphotoxin-deficient mice, the developing Flk-1+ white pulp vessels failed to organize or upregulate MAdCAM-1, leading to altered spatial rearrangement of both the white pulp endothelial cells and the smooth muscle actin-expressing cells. In vitro, MAdCAM-1 directed the reorganization of LTβ receptor+ endothelial cells grown on Matrigel. LTα1β2 also regulated the maintenance of both MAdCAM-1 expression and mature MS structure in adult mice, contributing importantly to normal trafficking of CD11b+ cells in response to bacterial antigens. Together, our studies demonstrate that LTα1β2 and LTβ receptor signals control proper development and maintenance of the mature MS structure and implicate MAdCAM-1 in the structuring of the MS endothelial cells that is important for the movement of immune cells within the spleen. © 2009 Elsevier Inc. All rights reserved.