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Pluripotent stem cells are endless sources for in vitro engineering human tissues for regenerative medicine. Extensive studies have demonstrated that transcription factors are the key to stem cell lineage commitment and differentiation efficacy. As the transcription factor profile varies depending on the cell type, global transcriptome analysis through RNA sequencing (RNAseq) has been a powerful tool for measuring and characterizing the success of stem cell differentiation. RNAseq has been utilized to comprehend how gene expression changes as cells differentiate and provide a guide to inducing cellular differentiation based on promoting the expression of specific genes. It has also been utilized to determine the specific cell type. This review highlights RNAseq techniques, tools for RNAseq data interpretation, RNAseq data analytic methods and their utilities, and transcriptomics-enabled human stem cell differentiation. In addition, the review outlines the potential benefits of the transcriptomics-aided discovery of intrinsic factors influencing stem cell lineage commitment, transcriptomics applied to disease physiology studies using patients’ induced pluripotent stem cell (iPSC)-derived cells for regenerative medicine, and the future outlook on the technology and its implementation.