Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated p21 transcription but PUMA and Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the p21 and PUMA promoters, RNA Pol II elongation was only observed along the PUMA gene and not the p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both p21 and PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic PUMA over anti-apoptotic p21 genes.