The progressive rise of mature CD5 super(+) B lymphocytes, despite the low proportion of proliferating cells, has led to the notion that B cell chronic lymphocytic leukemia (B-CLL) is primarily related to defective apoptosis. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo spontaneous apoptosis when cultured in vitro. To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk super(-), with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF- Kappa B activities. Interactions with fibroblasts sustained a basal level of Akt and NF- Kappa B activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the down-regulation of the prosurvival Bcl-2 family protein Bcl-x sub(L) and the caspase inhibitor proteins FLIP sub(L) and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF- Kappa B activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.