The p53 protein is a primary mediator of cellular apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has shown that the majority of childhood acute lymphoblastic leukemia (ALL) cases express a wild type p53 gene, although the functionality of the p53 pathway has rarely been validated. In the present study, the integrity of the p53 pathway was investigated in a panel of ALL cell lines and xenografts established from direct patient explants in immune-deficient mice. A focused real-time quantitative reverse transcription PCR array of known p53-regulated genes identified p21(WAF1) (CDKN1A) as the highest ranked gene to be differentially expressed between B-cell precursor (BCP)-ALL and T-ALL xenografts following exposure to the DNA-damaging drug etoposide. Lack of p21(WAF1) induction was observed in six of seven T-ALL xenograft lines, as well as primary T-ALL cells following irradiation exposure, despite an otherwise functional p53 response. Repression of p21(WAF1) in T-ALL cells was associated with decreased acetylated H3K9 localized at its promoter compared with BCP-ALL cells, together with increased CpG methylation within the first exon and intron. Although the histone deacetylase inhibitor vorinostat failed to induce p21(WAF1) in T-ALL samples, the combination of vorinostat and the demethylating agent decitabine reactivated expression of the silenced p21(WAF1) gene in the Molt-4 T-ALL cell line. Considering the known anti-apoptotic function of p21(WAF1), our findings have significant implications for the responses of T- versus BCP-ALL cells to chemotherapeutic drugs that induce p21(WAF1).