Signaling mediated by various Notch receptors and their ligands regulates diverse biological processes, including lymphoid cell fate decisions. Notch1 is required during T-cell development, while Notch2 and the Notch ligand Delta-like1 control marginal zone B (MZB) cell development. We previously determined that Mastermind-like (MAML) transcriptional coactivators are required for Notchinduced transcription by forming ternary nuclear complexes with Notch and the transcription factor CSL. The 3 MAML family members (MAML1-MAML3) are collectively essential for Notch activity in vivo, but whether individual MAMLs contribute to the specificity of Notch functions is unknown. Here, we addressed this question by studying lymphopoiesis in the absence of the Maml1 gene. Since Maml1^−/− mice suffered perinatal lethality, hematopoietic chimeras were generated with Maml1^−/−, Maml1^+/−, or wild-type fetal liver progenitors. Maml1 deficiency minimally affected T-cell development, but was required for the development of MZB cells, similar to the phenotype of Notch2 deficiency. Moreover, the number of MZB cells correlated with Maml1 gene dosage. Since all 3 Maml genes were expressed in MZB cells and their precursors, these results suggest that Maml1 is specifically required for Notch2 signaling in MZB cells.