Cell heterogeneity is a general feature of biological tissues. Standard transcriptome analysis approaches require tens of thousands of cells to provide an average view of gene expression and ignore the information of gene expression heterogeneity. The single-cell RNA-Seq technologies profile gene expression at the single-cell level and serve as powerful tools to identify distinct phenotypic cell types within a heterogeneous population. The single-cell RNA-Seq technologies have been developed rapidly in recent years. The methodological progress includes a variety of cDNA amplification methods, the quantitative analysis of the sensitivity and noise of the technologies, and the development of the low-coverage high-throughput single-cell RNA-Seq and the in situ RNA-Seq technologies. Furthermore, the scope of application is extended from early embryonic development to tissue and organ development, immunology and oncology. In this review, we discuss recent progress in methodology and applications of the single-cell RNA-Seq technologies.