Malignant B cells from chronic lymphocytic leukemia (B-CLL) patients have a long survival in vivo, although, in culture, they spontaneously die by apoptosis. Here, we analyzed the capacity of accessory leukocytes to modulate apoptosis of B-CLL cells in vitro. To this end, we performed long-term cultures using total mononuclear cells (TMC) from B-CLL patients and TMC depleted from monocytes, NK cells and T lymphocytes (B-CLL cells). In all the patients studied (n = 25) the presence of accessory leukocytes markedly prolonged the survival of B-CLL cells. The anti-apoptotic effect was exerted by monocytes and, to a lesser degree, NK cells, partially through the release of soluble factors. Indeed, accessory leukocytes separated from leukemic cells by semipermeable membranes were still able to prolong B-CLL cell survival. By flow cytometric analysis we found that the protective effect of non-malignant cells was associated with delayed down-regulation of Bcl-2 expression on leukemic cells. By contrast, the expression of Fas and Fas ligand proteins was unchanged in most samples. Our findings suggest that monocytes and NK cells, by delaying leukemic cell apoptosis, may play a role in B-CLL cell accumulation in vivo.