Altering chromatin structure by blocking histone deacetylase activity with specific inhibitors such as trichostatin A can result in an up-regulation of gene expression. In this report, however, we show that expression of the ETS domain transcription factor PU.1 is down-regulated in cells following the addition of trichostatin A. The loss of PU.1 is seen at both the mRNA and protein levels in multiple cell lines and is reversible following removal of the drug. More importantly, we show that the loss of PU.1 results in a loss of PU.1 target gene expression, including CD11b, c-fms, Toll-like receptor 4, and scavenger receptor. Chromatin immunoprecipitation analysis of cells treated with trichostatin A showed a significant increase in the acetylation of histone H4, but not histone H3, across approximately 650 bp of the PU.1 promoter region. Our data suggest that the consequences of using drugs that inhibit histone deacetylase activity may be a loss of blood cell development and/or function due to a block in PU.1 gene expression.