The Escherichia coli drug efflux network is highly complex and comprises 35 drug efflux pumps exhibiting varying degrees of functional redundancy, which has limited the study of efflux pump function. Harnessing a recently developed efflux platform built upon a highly efflux-deficient mutant background, EKO-35, I determined the substrate profiles of each individual drug efflux pump within the E. coli drug efflux network and summarized the physicochemical properties affecting the transport of compounds across the outer membrane (OM). Surprisingly, loss of 35 inner membrane drug efflux pumps affected the susceptibility of E. coli to several poorly-characterized synthetic antimicrobial agents to a lesser extent than loss of the major OM channel TolC. This channel forms complexes with numerous multidrug-resistant efflux pumps to facilitate the export of compounds across the OM. While investigating the basis of these susceptibility differences, I revealed that one of these synthetic compounds is an inhibitor of MsbA, an essential transporter involved in OM biogenesis. The genetic repression of msbA in a ∆tolC mutant resulted in increased sensitivity to this compound, which subsequently identified a synthetic genetic interaction between MsbA and TolC. I also showed that synthetic genetic interactions with TolC extend to other essential cell envelope biogenesis components. Overall, this thesis provides important insight into the physicochemical properties underlying the transport of drugs across the E. coli cell envelope, and reveals the potential involvement of TolC in cell envelope biogenesis and maintenance.