The ascent of antibiotic resistance poses a serious threat to human health. Left unaddressed, we could enter a “post-antibiotic” era where everyday infections prove fatal. As such, new ways of treating these infections or prolonging current antibiotic efficacy are needed. One method for prolonging antibiotic efficacy is to design “anti-evolution” co-therapies that target and inhibit the primary resistance pathway evolved against the antibiotic. Administering this co-drug alongside the antibiotic could then delay the onset of resistance. Previously, our lab found that the clinically important pathogen, Enterococcus faecalis, evolved resistance to the rescue-drug, daptomycin (DAP), via activating mutations to the LiaFSR membrane-stress-response pathway and cardiolipin synthase (cls) that redistribute membrane phospholipids and divert DAP binding away from vulnerable septal targets. While LiaFSR is heavily implicated in Enterococcus faecium DAP resistance, several clinical isolates containing activated liaFSR alleles are DAP tolerant and do not display the redistribution phenotype; similarly, several DAP resistant E. faecium isolates possess mutations in alternative pathways to LiaFSR. To identify the prominent DAP resistance mechanisms in E. faecium and further understand the role of LiaFSR, this thesis evaluated how clinical E. faecium evolved DAP resistance A) in the presence of activated LiaFSR alleles, and B) when liaR was deleted from the genome. We found that unlike E. faecalis, E. faecium evolved multiple and different resistance strategies that were heavily influenced by the environment within which they were adapted and that DAP repulsion from the cell surface was the more common mode of resistance. We also found that deletion of liaR significantly delayed the onset of resistance and that the resulting mechanisms were quite complex and varied, suggesting that this deletion significantly hindered the bacterial population which then struggled to find adaptive mutations. Regardless of environment or the presence of liaR, evolution converged on mutations to cls, highlighting the importance of membrane modification in enterococcal DAP resistance. While E. faecium can use multiple pathways to resist DAP, the increased timeline to DAP resistance when liaR is deleted supports the hypothesis for developing a small molecule inhibitor of this system to increase DAP efficacy against enterococcal infections.