AbstractCyclic adenosine monophosphate response element binding (CREB)–binding protein (CBP) and p300 are multidomain transcriptional coactivators that help assemble large regulatory complexes at sites of active transcription. Nullizygosity of CBP or p300 results in pervasive defects in hematopoiesis. To systematically assess the structural domains of p300 required for normal hematopoiesis, we used recombinase-mediated cassette exchange to create an allelic series of coisogenic embryonic stem cells, each expressing a different mutant of p300 from the endogenous locus. We found that deletion of either the KIX or CH1 domain caused profound and pervasive defects in hematopoiesis, whereas the loss of most other domains had only lineage-restricted effects. When expressed from the p300 locus, an extra copy of CBP largely compensated for a lack of p300. Surprisingly, mutation of the p300 histone acetyltransferase (HAT) domain had minimal effects on hematopoiesis, and actually increased progenitor and stem cell numbers and proliferative potential. Our results suggest that, in distinct contrast to other organ systems, HAT activity does not provide a critical function for hematopoietic development and emphasizes the importance of enzyme-independent functions of p300.