Host proteins, termed host dependency factors (HDFs), are crucial for productive HIV infection but dispensable for cellular viability, thereby representing promising therapeutic targets. Here, Park et al. conduct a CRISPR-based genetic screen in a CD4+ T cell line to identify five HDFs (the HIV co-receptors CD4 and CCR5, as well as TPST2, SLC35B2, and ALCAM) that, when inactivated, conferred protection from HIV infection. Using the CD4+ T cell line and primary CD4+ T cells, TPST2 and SLC35B2 were shown to facilitate CCR5 recognition by the HIV envelope, whereas ALCAM mediated cell aggregation, which is required for cell-to-cell HIV transmission.