The Mesp1 lineage contributes to cardiac, hematopoietic and skeletal myogenic development. Interestingly, muscle stem cells residing in craniofacial skeletal muscles primarily arise from Mesp1+ progenitors, but those in trunk and limb skeletal muscles do not. To gain insights into the difference between the head and trunk/limb muscle developmental processes, we studied Mesp1+ skeletal myogenic derivatives via single-cell RNA-seq and other strategies. Using a doxycycline-inducible Mesplexpressing mouse embryonic stem cell line, we found that the development of Mespl-induced skeletal myogenic progenitors can be characterized by dynamic expression of PDGER alpha and VCAM1. Single-cell RNA-seq analysis further revealed the heterogeneous nature of these Mesp1+ derivatives, spanning pluripotent and mesodermal to mesenchymal and skeletal myogenic. We subsequently reconstructed the single-cell trajectories of these subpopulations. Our data thereby provide a cell fate projection of Mesp1-induced skeletal myogenesis. (C) 2019 Elsevier Inc. All rights reserved.