T-lymphocyte migration under flow is critical for immune responses, but the mechanisms by which flow modulates the migratory behaviors of T-lymphocytes remain unclear. Human peripheral blood T-lymphocytes (PBTLs), when stimulated with phorbol 12-myristate 13-acetate (PMA), stretched their cell bodies dramatically and moved along the flow direction. In contrast, stromal cell-derived factor-1α-stimulated PBTLs deformed and migrated in a random manner. Here we elucidated the molecular mechanisms underlying flow-induced directionality and deformation of PMA-stimulated PBTLs. PMA primed PBTLs for polarization under flow, with protein kinase C (PKC)-δ enriched in the leading edge, PKC-βI in the microtubule organizing center, and PKC-βII in the uropod and peripheral region. PKC-δ regulated cell protrusions in the leading edge through Tiam1/Rac1/calmodulin, whereas PKC-β regulated RhoA/Rho-associated kinase activity and microtubule stability to modulate uropod contractility and detachment. Our findings indicate that PKC-δ and -β coordinate in the cell leading edge and uropod, respectively, to modulate the directionality and deformability of migratory T-lymphocytes under flow.