This chapter provides a review of recent advances in understanding the importance of alternative pre-messenger RNA splicing in stem cell biology. The majority of transcribed pre-mRNAs undergo RNA splicing where introns are excised and exons are juxtaposed to form mature messenger RNA sequences. This regulated, selective removal of whole or portions of exons by alternative splicing provides avenues for control of RNA abundance and proteome diversity. We discuss several examples of key alternative splicing events in stem cell biology and provide an overview of recently developed microarray and sequencing technologies that enable systematic and genome-wide assessment of the extent of alternative splicing during stem cell differentiation.