Emerging evidence suggests that the survival of B-cell chronic lymphocytic leukemia (CLL) cells is dependent on microenvironmental influences such as antigenic stimulation and support by stromal cells. Akt, also known as protein kinase B, is a central component in prosurvival signaling downstream of these events. We investigated the role of Akt and its modulation by the protooncogene T-cell leukemia 1a (Tcl1a) in the survival pathways of primary CLL samples and CLL-derived prolymphocytic cell lines MEC-1 and MEC-2. Akt activation was increased by the protective presence of human bone marrow stromal cells and B-cell receptor mimicking signals but antagonized by direct Akt blockade with the novel specific inhibitor AiX, with preferential apoptosis induction in CLL cells with an unmutated immunoglobulin status, which predicts poor clinical outcome. In addition, we found a direct interaction of Akt with Tcl1a in an endogenous coimmunoprecipitation assay. Confirming the critical role of Tcl1a in modulating Akt signaling, Akt activation was enhanced by overexpressing Tcl1a in CLL. In contrast, decreasing Tcl1a levels by small interfering RNA reduced Akt activation in the fludarabine-insensitive CLL cell line MEC-2 and sensitized the malignant cells to fludarabine treatment. In summary, our data reveal a significant role for the Akt-Tcl1a axis in CLL survival and propose a further evaluation of this interplay for targeting chemoresistance phenomena.