Glucocorticoids are keystone drugs in the treatment of childhood acute lymphoblastic leukemia (ALL). To get more insight in signal transduction pathways involved in glucocorticoid-induced apoptosis, Affymetrix U133A GeneChips were used to identify transcriptionally regulated genes on 3 and 8 hours of prednisolone exposure in leukemic cells of 13 children as compared with nonexposed cells. Following 3 hours of exposure no significant changes in gene expression could be identified. Following 8 hours of exposure, 51 genes were differentially expressed (P < .001 and false discovery rate < 10%) with 39 genes being up-regulated (median, 2.4-fold) and 12 genes were down-regulated (median, 1.7-fold). Twenty-one of those genes have not been identified before to be transcriptionally regulated by prednisolone. Two of the 3 most highly up-regulated genes were tumor suppressor genes, that is, thioredoxin-interacting protein (TXNIP; 3.7-fold) and zinc finger and BTB domain containing 16 (ZBTB16; 8.8-fold). About 50% of the differentially expressed genes were functionally categorized in 3 major routes, namely MAPK pathways (9 genes), NF-kappaB signaling (11 genes), and carbohydrate metabolism (5 genes). Biologic characterization of these genes and pathways might elucidate the action of glucocorticoids in ALL cells, possibly suggesting causes of glucocorticoid resistance and new potential targets for therapy.