Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.