Human induced pluripotent stem cells (hiPSC) possess the ability to differentiate into a multitude of tissue types but display heterogeneity in the propensity of differentiation into specific tissue lineages. An examination of isogenic hiPSC lines revealed variations in the endoderm propensity under directed differentiation conditions. Characterization of the transcriptome and proteome of the hiPSC lines showed that MIXL1 activity at the early differentiation stage correlated with the efficacy of generating definitive endoderm and further differentiation into endoderm derivatives. Enforced expression of MIXL1 in the endoderm-incompetent hiPSCs enhanced the propensity of endoderm differentiation, suggesting that modulation of key drivers of lineage differentiation can re-wire hiPSC to the desired lineage propensity for stem cell products.