The development of small-molecule activators of p53 is currently focused on malignancies containing a wild-type p53 genotype, which is present in most leukemias. JNJ-26854165 is one such p53-activating agent, but its mechanism of action remains to be elucidated. Here, we report the effects of JNJ-26854165 in acute leukemias. JNJ-26854165 treatment induced p53-mediated apoptosis in acute leukemia cells with wild-type p53, in which p53 rapidly drives transcription-independent apoptosis followed by activation of a transcription-dependent pathway. JNJ-26854165 accelerated the proteasome-mediated degradation of p21 and antagonized the transcriptional induction of p21 by p53. Interestingly, JNJ-26854165 induced S-phase delay and upregulated E2F1 expression in p53 mutant cells, resulting in apoptosis preferentially of S-phase cells. E2F1 knockdown blocked apoptosis induced by JNJ-26854165 in p53 mutant cells. Apoptotic activity of JNJ-26854165 against primary acute leukemia cells was maintained in leukemia/stroma cocultures, unlike doxorubicin, which has reduced cytrotoxicity in coculture systems. JNJ-26854165 synergizes with 1-β-arabinofuranosylcytosine or doxorubicin to induce p53-mediated apoptosis. Our data suggest that JNJ-26854165 may provide a novel therapeutic approach for the treatment of acute leukemias. The presence of p53-independent apoptotic activity in addition to p53-mediated apoptosis induction, if operational in vivo, may prevent the selection of p53 mutant subclones during therapy.