Trithorax group (TrxG) proteins play critical roles in transcriptional activation by promoting methylation of histone H3 Lysine 4 (H3K4), but the precise functions of the individual TrxG members during embryonic differentiation are not fully understood. Here we show that Mll2, a TrxG member, is required for proliferation but is dispensable for maintaining the pluripotency of mouse embryonic stem cells (ESCs). In addition, differentiation of ESCs toward mesodermal and endodermal lineages is severely altered and, in particular, the cardiac lineage differentiation of ESCs is completely abolished in the absence of Mll2. Moreover, the expression of core cardiac transcription factors and the levels of H3K4 tri-methylation of these cardiac-specific promoters are significantly decreased by the loss of Mll2. Taken together, our results reveal a critical role for Mll2 in proliferation and cardiac lineage differentiation of mouse ESCs, and provide novel molecular insight into the mechanisms of cardiac development and disease.