The Ikaros family of proteins are DNA binding factors required for correct development of B and T lymphocytes. Cytogenetic studies have shown that these proteins form complexes with pericentromeric heterochromatin in B cells, and the colocalization of transcriptionally silent genes with these complexes suggests that Ikaros could silence transcription by recruiting genes to heterochromatin. Here we show that a site in the λ5 promoter that binds Ikaros and Aiolos is required for silencing of λ5 expression in activated mature B cells. Analysis of methylation and nuclease accessibility indicates that the silenced λ5 gene is not heterochromatinized in B cells, despite being associated with pericentromeric heterochromatin clusters. We also found that a promoter mutation, which affects Ikaros-mediated silencing of λ5 expression, is not rescued in a transgenic line that has the gene integrated into pericentromeric heterochromatin. Our results indicate that the Ikaros proteins initiate silencing of λ5 expression through a direct effect on the promoter with localization to pericentromeric heterochromatin likely to affect the action of Ikaros on regulatory sequences rather than causing heterochromatinization of the gene.