In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles. The tyrosine protein kinase Lyn, is aberrantly expressed in the malignant and stromal cells in CLL tissue. We therefore studied the role of Lyn in the EV-based communication and tumor support. We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient and deficient stromal cells and primary CLL cells. Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient ones. Also, they induced stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and deficient stromal cell highlighted 72 significantly differentially expressed proteins, many of which belonging to the extracellular matrix organization, such as collagen, nidogen, fibronectin and endosialin (CD248). CD248, a marker of certain tumors and of cancer associated fibroblast (CAF) was significantly depleted in Lyn-deficient HS-5 cells. A knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cells survival feeding capacity compared to wildtype or scrambled control cells. The presented data provide preclinical evidence, that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising the EV release and their concentration of functional molecules, such as endosialin.