To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.