Abstract Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL−/−) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL−/− B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration.