Although Activin/Nodal signaling regulates pluripotency of human embryonic stem (ES) cells, how this signaling acts in mouse ES cells remains largely unclear. To investigate this, we confirmed that mouse ES cells possess active Smad2-mediated Activin/Nodal signaling and found that Smad2-mediated Activin/Nodal signaling is dispensable for self-renewal maintenance but is required for proper differentiation toward the mesendoderm lineage. To gain insights into the underlying mechanisms, Smad2-associated genes were identified by genome-wide chromatin immunoprecipitation-chip analysis. The results showed that there is a transcriptional correlation between Smad2 binding and Activin/Nodal signaling modulation, and that the development-related genes were enriched among the Smad2-bound targets. We further identified Tapbp as a key player in mesendoderm differentiation of mouse ES cells acting downstream of the Activin/Nodal-Smad2 pathway. Taken together, our findings suggest that Smad2-mediated Activin/Nodal signaling orchestrates mesendoderm lineage commitment of mouse ES cells through direct modulation of corresponding developmental regulator expression.