Abstract Background: Prenatal immune development may play an important role in the etiology of childhood acute lymphoblastic leukemia (ALL). Methods: Seven cytokines, IL1β, IL4, IL6, IL8, GM-CSF, TNFα, and VEGF, were analyzed in blood spots collected at birth from 1,020 ALL cases and 1,003 controls participating in the California Childhood Leukemia Study. ORs and 95% confidence intervals (95% CI) associated with an interquartile range increment in cytokine levels were calculated using logistic regression, adjusting for sociodemographic and birth characteristics. Results: We found that patients with ALL were born with higher levels of a group of correlated cytokines than controls [IL1β: OR of 1.18 (95% confidence interval [CI], 1.03–1.35); IL8: 1.19 (1.03–1.38); TNFα: 1.15 (1.01–1.30); VEGF: 1.16 (1.01–1.33)], especially among children of Latina mothers (ORs from 1.31 to 1.40) and for ALL with high hyperdiploidy (ORs as high as 1.27). We found that neonatal cytokine levels were correlated with neonatal levels of endogenous metabolites which had been previously associated with ALL risk; however, there was no evidence that the cytokines were mediating the relationship between these metabolites and ALL risk. Conclusions: We posit that children born with altered cytokine levels are set on a trajectory towards an increased risk for subsequent aberrant immune reactions that can initiate ALL. Impact: This is the first study to evaluate the interplay between levels of immunomodulatory cytokines at birth, prenatal exposures, and the risk of childhood ALL.