RNA interference (RNAi) is a novel phenomenon that can induce post-transcriptional gene silencing (PTGS) both in animals and plants. RNAi is effective in suppressing specific gene expression in the early mouse embryonic cells and in undifferentiated embryonic stem (ES) cells. In this study, we demonstrate that dsRNA is effective in inducing PTGS in differentiated ES cells: CD34+ embryoid body (EB) cells, as confirmed by western blot and immunocytochemical staining. PU.1 is a key transcription factor in myeloid differentiation. Undifferentiated ES cells do not express PU.1; however it is expressed when ES cells differentiate into EBs. PU.1 could be suppressed by the specific PU.1 dsRNA, but not non-specific Lamin A/C dsRNA, in the CD34+ EB cells when they were induced to myeloid differentiation in the presence of GM-CSF and IL-3. As a consequence, the level of expression of CD115 (M-CSF receptor), one of the downstream genes regulated by PU.1 is decreased in PU.1 dsRNA treated CD34+ EB cells, but not in Lamin A/C dsRNA treated cells. To explore this phenomenon in other myeloid gene, we also found that C/EBPalpha gene could be knocked down by C/EBPalpha dsRNA. Our finding demonstrates that RNAi is effective in inhibiting specific gene expression in differentiated ES cells. RNAi offers a new methodology for study of hematopoietic regulation using ES cell differentiation.