Pervasive transcription has been reported in many eukaryotic organisms, revealing a highly interleaved transcriptome organization that involves thousands of coding and non‐coding RNAs. However, to date, the biological impact of transcriptome complexity is still poorly understood. Here I will review how subtle variations of the transcriptome can lead to divergent cellular phenotypes by fine‐tuning both its coding potential and regulation. I will discuss strategies that can be used to link molecular variations with divergent biological outcomes. Finally, I will explore the implication of transcriptional complexity for our understanding of gene expression in the context of cell‐to‐cell phenotypic variability. Copyright © 2017 John Wiley & Sons, Ltd.