The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. TheE2Agene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved φ-x-x-φ-φ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved φ-x-x-φ-φ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.