Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19Arf, a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19Arf and a second tumor suppressor, p16Ink4a, that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16Ink4a plays a role in Ab-MLV transformation, primary transformants derived from Arf-/- and p16Ink4a-/- mice were compared. A fraction of those derived from Arf-/- animals underwent crisis, and even though all p16Ink4a-/- primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf-/- cells infected with a virus that expresses both v-Abl and p16Ink4a revealed that p16Ink4a expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16Ink4a can suppress transformation by inducing apoptosis.