HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6Gag and EIAV p9Gag proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.