Abstract Body: The differentiation of cardiomyocytes (CMs) from non-myocyte progenitors is accompanied by profound shifts in mitochondrial morphology and energetics. Mitochondria change shape through processes of fusion (joining) and fission (dividing) of their outer and inner mitochondrial membranes (OMM and IMM, respectively). Cristae, folds in the IMM, are major sites of oxidative energy production. More fused networks with denser cristae tend to be more energetically efficient. Compared to pluripotent stem cells (PSCs), CMs have higher mitochondrial mass arranged in larger networks with denser cristae. These structural changes support the higher energetic activity required for CM function. Despite this, little work has investigated the contributions of fission and fusion regulators to CM differentiation and maturation. We recently showed that loss of mitochondrial fission regulator 1-like protein (MTFR1L) increased conversion of cardiac fibroblasts (CFs) to functional induced CMs. However, expression of Mtfr1l in native CMs is high relative to other cell types, including CFs. We thus hypothesized that MTFR1L regulation of mitochondrial morphology and cristae organization is a barrier to CM differentiation from non-myocytes but promotes maturation and maintenance of CM identity. We evaluated expression of Mtfr1l and other fusion and fission regulators at multiple time points during CM differentiation from murine and human PSCs. We then characterized mitochondrial morphology and energetics using metabolic flux assays with confocal and electron microscopy. Finally, we evaluated CM differentiation efficiency and functional maturity in cells expressing control and Mtfr1l-targeting shRNAs. We found that loss of Mtfr1l promotes mitochondrial energetics and CM differentiation. We also report a novel role for Mtfr1l in maintenance of CM identity. Considering our previous results in direct cardiac reprogramming, we suggest that MTFR1L activity may reduce cell fate plasticity in diverse cell types. In conclusion, the metabolic shift required for CM differentiation is facilitated by mitochondrial biogenesis, fusion and cristae reorganization. The fission promoter Mtfr1l is a barrier to this shift but plays a role in CM maturation and maintenance.