Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for blood progenitor cells is whether to adopt the lymphoid or myeloid fate. Previous work had shown that myocyte enhancer factor 2C (MEF2C) is indispensable for the lymphoid fate decision, yet the specific mechanism of action remained unclear. Here, we have identified early B cell factor-1 (EBF1) as a co-regulator of gene expression with MEF2C. A genome-wide survey of MEF2C and EBF1 binding sites identified a subset of B cell-specific genes that they target. We also determined that the p38 MAPK pathway activates MEF2C to drive B cell differentiation. Mef2c knockout mice showed reduced B lymphoid-specific gene expression as well as increased myeloid gene expression, consistent with MEF2C's role as a lineage fate regulator. This is further supported by interaction between MEF2C and the histone deacetylase, HDAC7, revealing a likely mechanism to repress the myeloid transcription program. This study thus elucidates both activation and repression mechanisms, identifies regulatory partners, and downstream targets by which MEF2C regulates lymphoid-specific differentiation.