The chimeric monoclonal antibody Rituximab is standard of care for patients suffering from B-cell Non-Hodgkin’s lymphoma (B-NHL). Rituximab mediates complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of CD20-positive human B cells. In addition, Rituximab sensitizes B-NHL cells to cytotoxic chemotherapy, and has direct apoptotic and antiproliferative effects. While expression of the CD20 antigen is a natural prerequisite for Rituximab sensitivity, cell-autonomous factors determining the response of B-NHL to Rituximab are less defined. To this end we have studied Rituximab-induced apoptosis in human B-NHL models. We find that Rituximab directly triggers apoptosis via the mitochondrial pathway of caspase activation. Expression of anti-apoptotic Bcl-xL confers resistance against Rituximab-induced apoptosis in vitro, and Rituximab treatment of xenografted B-NHL in vivo. B-NHL cells insensitive to Rituximab-induced apoptosis exhibit increased endogenous expression of multiple anti-apoptotic Bcl-2 family proteins, or activation of phosphatidylinositol-3-kinase (PI3K) signaling resulting in upregulation of Mcl-1. The former resistance pattern is overcome by treatment with the BH3-mimetic ABT-737, the latter by combining Rituximab with pharmacologic PI3K inhibitors. In conclusion, sensitivity of B-NHL cells to Rituximab-induced apoptosis is determined at the level of mitochondria. Pharmacologic modulation of Bcl-2 family proteins or their upstream regulators is a promising strategy to overcome Rituximab resistance.