Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols Total XIIIA (n 5 154), Total XV (n 5 498), and Total XVI (n 5 271), or Children's Oncology Group protocols POG 9906 (n 5 222) and AALL0232 (n 5 2163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome BeadChip array. In multivariate logistic regression, the intronic rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) had the strongest association with hypersensitivity (P 5 4.1 3 10-8 ; odds ratio [OR] 5 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruba HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2 expression compared with noncarriers (P 5 1.1 3 10-3 and 0.03, respectively). The top ranked nonsynonymous polymorphism was rs17885382 in HLA-DRB1 (P 5 3.2 3 10-6 ;OR5 1.63), which is in near complete linkage disequilibrium with the HLA-DRB107:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.