Hematopoiesis is regulated by a complex gene expression program. To gain further insight into the molecular mechanisms underlying this process in humans, we sampled the transcriptional activity of the CD34+ hematopoietic progenitor line KG1a by single-pass sequencing the 5' ends of 1018 clones from a unidirectional cDNA library. Searches of public databases with the resulting expressed sequence tags (ESTs) identified 101 clones that showed no sequence similarity to any of the existing entries and that were therefore considered to derive from previously undescribed genes. Of the remaining 917 ESTs, 553 (a total of 485 distinct transcripts) corresponded to known genes. A further 279 KG1a ESTs matched or exhibited sequence similarity to ESTs or genomic sequences from humans and other species. Among the latter were putative human orthologs of developmental and cell cycle control genes from Caenorhabditis elegans, Drosophila, and yeast, as well as genes whose predicted amino acid sequences showed similarity to mammalian transcription factors. Hybridization of selected novel KG1a ESTs to globally amplified cDNAs prepared from single primary human hematopoietic precursors and homogeneous populations of terminally maturing hematopoietic cells revealed transcripts that are expressed preferentially at a specific stage or in a particular lineage within the hematopoietic hierarchy. Thus, included in the KG1a EST dataset are candidates for new human genes that may play roles in hematopoietic differentiative progression and lineage commitment.