Activation of lymphocyte function-associated antigen-1 (LFA-1) by chemokines is fine-tuned by inside-out signaling mechanisms responsible for integrin-mediated adhesion modulation. In the present study, we investigated the possibility of qualitative variability of signaling mechanisms controlling LFA-1 activation in chronic lymphocytic leukemia (CLL) cells. We pursued a multiplexed comparative analysis of the role of the recently described chemokine-triggered rho-signaling module in human normal versus CLL B-lymphocytes. We found that the rho-module of LFA-1 affinity triggering is functionally conserved in normal B-lymphocytes. In contrast, in malignant B-lymphocytes isolated from patients with B-CLL, the role of the rho-module was not maintained, showing remarkable differences and variability. Specifically, RhoA and phospholipase D1 were crucially involved in LFA-1 affinity triggering by CXCL12 in all analyzed patients. In contrast, Rac1 and CDC42 involvement displayed a consistent patient-by-patient variability, with a group of patients showing LFA-1 affinity modulation totally independent of Rac1 and CDC42 signaling activity. Finally, phosphatidylinositol-4-phosphate 5-kinase isoform 1gamma (PIP5KC) was found without any regulatory role in all patients. The data imply that the neoplastic progression may completely bypass the regulatory role of Rac1, CDC42, and PIP5KC, and show a profound divergence in the signaling mechanisms controlling integrin activation in normal versus neoplastic lymphocytes, suggesting that patients with CLL can be more accurately evaluated on the basis of the analysis of signaling mechanisms controlling integrin activation. Our findings could potentially affect the diagnosis, prognosis, and therapy of CLL disorders.