Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.