The Ikaros family transcription factors (Ikaros, Helios, Aiolos, Eos) are major regulators of the development and function of immune cells. They are able to form all combinations of homo- and heterodimers. However, how the various dimers contribute to the regulation of gene expression is not known. We compared the function of the different dimers of Ikaros and Aiolos during pre-B cell differentiation. We found that each of the three possible dimers regulates both overlapping and specific sets of target genes. While the Aiolos containing dimers (homo and hetero) display a very broad target gene repertoire, the Ikaros homodimers bind and regulate much fewer genes. Indeed, Aiolos homodimers bind also to a larger set of GGAA-containing sequences in vitro, a feature which is mainly determined by one single amino acid difference in the DNA-binding domain when compared to Ikaros. We also showed that pre-B cell differentiation is orchestrated by a sequential function of the different dimers. Transition from pro-B to large pre-B cells, when Aiolos expression is barely detectable, is driven by Ikaros dimers, which mainly act as repressors targeting pathways for IL-7 signaling, cell adhesion and migration. As Aiolos expression increases in small pre-B and immature B cells, the Ikaros-Aiolos heterodimer and the Aiolos homodimer become respectively the predominant regulators of the transitions from the large pre-B to the small pre-B, and the small pre-B to the immature B cell stages, to control functions such as cell cycle, B cell receptor signaling or immune effector activation. Thus, our data reveal unexpected specificity among Ikaros family dimers, and highlight a well coordinated function of various family members to establish unique gene expression programs during immune cell differentiation.