There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL) but to date only static in vitro cultures have been used to study these phenomena. To address this we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L, CXCR4 (both P<0.0001), CD49d and CD5 (both P=0.003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the 'extravascular' space (EVS) (mean migration: 1.37% ± 2.14%, n=21). Migrated CLL cells had significantly higher expression of CD49d (P=0.02), MMP-9 (P=0.004), CD38 (P=0.009), CD80 (P=0.04) and CD69 (P=0.04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r(2)=0.47, P=0.01) and treatment with the CD49d blocking antibody, Natalizumab resulted in significantly decreased migration (P=0.01). Taken together our data provide evidence for a novel, dynamic and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL.