HIV Env undergoes conformational changes that drive virus entry and frustrate efforts to create stable immunogens. gp41 peptide fusion inhibitors interfere with conformational changes in Env in a dominant-negative manner and serve as tools to study Env conformations. Previously we found 2 genetic pathways of gp120 and gp41 mutations that confer cross-resistance to N and C heptad repeat peptide fusion inhibitors. Each pathway is defined by a key mutation in either the N or C heptad repeat of gp41 with accompanying gp120 mutations in CD4 binding site or V3 regions, respectively. Phenotypic studies show that mutations in the N heptad repeat greatly enhance susceptibility to sCD4 inhibition, while both pathways confer delayed entry kinetics. Sensitivity studies with a panel of monoclonals further reveal that mutations from both pathways favor Envs with more open or relaxed conformations. These studies identify residues in gp41 and gp120 that functionally interact to simultaneously influence receptor use, entry kinetics, and openness of Env conformation. The findings further highlight functional consequences of mutations that confer a more open Env structure.