While many sequence-specific transcription factors (TFs) have been identified as key regulators of hematopoietic stem cell (HSC) lineage determination, the function of general TFs in HSC behavior is poorly understood. To evaluate the function of the TFIID subunit TAF1 in normal hematopoiesis, we generated Taf1 conditional knockout (cKO) mice and identified an essential role of TAF1 in fetal hematopoiesis. Surprisingly, TAF1 deletion in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of the hematopoietic stem and progenitor cell (HSPC) compartment, with increased self-renewal and impaired differentiation capacity of these cells. TAF1-null HSPCs failed to produce mature blood cells in chimeric mice; these cells also failed to upregulate key differentiation genes when induced to differentiate in vitro. TAF1 loss not only disrupted TFIID chromatin recruitment but also reduced RNA polymerase II (RNAPII) promoter-proximal pausing. Thus, HSPCs utilize distinct transcriptional regulatory mechanisms to undergo differentiation versus maintaining self-renewal.