Numerous studies have documented the efficacy of rituximab (either alone and/or in combination with drugs) in eradicating tumor cells in vitro and in vivo. However, a subset of patients does not respond or relapse after initial rituximab treatment. In an attempt to recapitulate the non-responders and/or relapsed situations, rituximab-refractory B-NHL Ramos and Daudi clones were generated by growing the cells in the presence of step-wise increasing concentrations of rituximab followed by multiple rounds of limiting dilution assay. Single cells were then propagated as clones and assayed for cellular and molecular alterations when compared to the parental cell lines. This study investigates two rituximab-resistant clones, namely, Ramos RR1 and Daudi RR1. A partial decrease in surface CD20 expression, namely, decreased MFI compared to parental cells was observed in the clones. Both clones exhibited resistance to complement-dependent cytotoxicity (CDC). Rituximab was incapable of either inhibiting cellular growth or inducing apoptosis (both in monomeric and cross-linked forms). The clones developed significant cross-resistance to chemotherapeutic drugs and importantly, rituximab had lost its chemo-sensitizing effect on these clones. Detailed analysis revealed a hyper-activated status of the ERK1/2 and NF-κB signaling pathways and rituximab was inefficient in inhibiting these pathways in the clones. Strikingly, selective over-expression of Bcl-xL and the exhibition of higher resistance to a wide array of anti-neoplastic agents were observed in the clones concordant with the protective role of Bcl-xL. However, pharmacological inhibition of the NF-κB and the ERK 1/2 pathways as well as the proteasome inhibitor bortezomib (Velcade) efficiently sensitized the clones to structurally and functionally distinct drugs including topoisomerase II inhibitor, DNA damaging agents, and microtubule poisons, albeit to varying degrees. The above inhibitors also reduced Bcl-xL levels further confirming the pivotal role played by Bcl-xL is clone resistance. The generation of rituximab-resistant clones provides novel means to further investigate the underlying mechanisms that may govern rituximab resistance in patients. Further, these results provide a rational molecular basis for the utilization of pharmacological inhibitors of constitutive signaling pathways in combination with cytotoxic stimuli in the treatment of rituximab-refractory NHL cells.