Leukocytes travel between different tissues, ranging from an inflamed site, bone marrow to lymph node after hematopoiesis for further development and maturation, to another tissue, through the vasculature. To efficiently reach the destination, circulating leukocytes in the blood stream must be able to adhere and exit the circulation. Recently, T lymphocytes have been shown to distinguish the direction of shear flow and migrate in a direction that is determined by adhesion molecules on the surface. T lymphocytes with LFA-1-ICAM-1 interaction migrate upstream (i.e. in the opposite direction of shear flow), and those with VLA-4-VCAM-1 binding migrate downstream (i.e. with the direction of flow). In the first aim, we study the crosstalk of LFA-1 and VLA-4 integrins in the upstream migration. We showed that while LFA-1 and VLA-4 mediated migration independently show very different directional response, LFA-1 and VLA-4 together potentiates the upstream migration during and post flow. The persistent direction of migration is mediated by PI3K. We uncover the intracellular signaling mechanism responsible for the upstream migration of CD4+ T lymphocytes with mice models in collaboration with Dr. Janis K. Burkhardt at Children’s Hospital of Philadelphia. We found that, downstream of LFA-1, Crk and c-Cbl are key in the upstream direction. Lastly, in the third aim we investigate how the substrate mechanics affect the motility of CD4+ T lymphocytes under static and flow conditions. Specifically, we reported that while LFA-1-mediated migration under static conditions showed stiffness-dependent motility, the upstream migration under flow was unaffected. Together, we provide more insight to understanding the mechanism of the upstream migration.