Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell-derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle-isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell-derived cardiomyocytes. Stem cell-derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell-derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell-derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.