Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G(1)-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G(1)-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G(1)-S transition is controlled by phosphorylation in response to signaling events that down-modulate its DNA binding activity.