Etoposide is used to treat a wide-range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric-AML patients become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric AML patients treated with etoposide containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.