Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.