The immune system has diverse types of cells that are differentiated or activated via various signaling pathways and transcriptional regulation upon challenging conditions. Immunophenotyping by flow and mass cytometry are the major approaches for identifying key signaling molecules and transcription factors directing the transition between the functional states of immune cells. However, few proteins can be evaluated by flow cytometry in a single experiment, preventing researchers from obtaining a comprehensive picture of the molecular programs involved in immune cell differentiation. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled unbiased genome-wide quantification of gene expression in individual cells on a large scale, providing a new and versatile analytical pipeline for studying immune cell differentiation.