Phosphorylation of H2AX on Ser139 (H2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called H2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous H2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21(WAF1) (p21) exhibit low numbers of H2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21-/-) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21-/- and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between H2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous H2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of H2AX.