Hematopoietic stem cells (HSCs) maintain tissue homeostasis by rapidly responding to environmental changes. Although this function is well understood, the molecular mechanisms governing this characteristic are largely unknown. We used a sequenced-based strategy to explore the role of both transcriptional and post-transcriptional regulation in HSC biology. We characterized the gene expression differences between HSCs, both quiescent and proliferating, and their differentiated progeny. This analysis revealed a large fraction of sequence tags aligned to intronic sequences, which we showed were derived from unspliced transcripts. A comparison of the biological properties of the observed spliced versus unspliced transcripts in HSCs showed that the unspliced transcripts were enriched in genes involved in DNA binding and RNA processing. In addition, levels of unspliced message decreased in a transcriptspecific fashion after HSC activation in vivo. This change in unspliced transcript level coordinated with increases in gene expression of splicing machinery components. Combined, these results suggest that post-transcriptional regulation is important in HSC activation in vivo. STEM CELLS 2006;24:662-670