Here we show that EGF and EGF receptor (EGFR) are trafficked through a subpopulation of multivesicular endosomes/bodies (MVBs) that are distinct from morphologically identical vacuoles that label for the late endosomal marker lyso-bisphosphatidic acid (LBPA). EGF stimulation increases both MVB biogenesis and inward vesiculation within EGFR-containing MVBs. Deletion of annexin 1, a substrate of EGFR tyrosine kinase, abolishes the effect of EGF stimulation on inward vesiculation. This phenotype is reversible by transfection with wild-type but not Y21F phosphorylation mutant annexin 1. Deletion of annexin 1 has no effect on EGF-stimulated MVB biogenesis, suggesting that MVB biogenesis and inward vesiculation within MVB are mediated by separate mechanisms. Loss or depletion of annexin 1 has no effect on EGF degradation and causes only a small delay in EGFR degradation, indicating that annexin 1 operates downstream of Hrs- and ESCRT-mediated sorting and is required solely for EGF-stimulated inward vesiculation. Annexin 1 accumulates on internal vesicles of MVB after EGF-stimulated inward vesiculation, suggesting that it may be required for a late stage in inward vesiculation.