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Venetoclax inhibits the pro-survival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL, but with eventual loss of efficacy. A spectrum of sub-clonal genetic changes associated with venetoclax resistance have now been described. To fully understand clinical resistance to venetoclax, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired venetoclax resistance. These appear to be multi-layered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the pro-survival family members. This includes previously described mutations in BCL2and amplification of the MCL1gene but are heterogeneous across and within individual patient's leukemias. Changes in the pro-apoptotic genes are notably uncommon, except for single cases with sub-clonal losses of BAXor NOXA. Much more prominent was universal MCL1gene upregulation. This was driven by an overlying layer of emergent NF-κB activation which persisted in circulating cells during venetoclax therapy. We discovered that MCL1could be a direct transcriptional target of NF-κB. Both the switch to alternative pro-survival factors and NF-κB activation largely dissipate following venetoclax discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade venetoclax-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent venetoclax resistance and provide a specific biological justification for the strategy of venetoclax discontinuation once maximal response is achieved rather than maintaining long-term selective pressure with the drug.