Sequential up- and down-regulation of a handful of critical transcription factors is required for proper neutrophil differentiation. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML) and specific granule deficiency. In order to understand the molecular background for normal and malignant granulopoiesis, a good model system is required that faithfully mimics the in vivo transcription factor expression profiles. The two human leukemic cell lines HL60 and NB4 have been widely used as model cell lines for these purposes. Differentiation of HL60 and NB4 cells resulted in asynchronous differentiation to morphologically mature neutrophils over a period of 5-7 days. To obtain cell populations of more even maturity, cells at different stages of in vitro differentiation were purified by immunomagnetic isolation. This resulted in three cell populations that could be classified as promyelocytes, myelocytes/metamyelocytes, and mature neutrophils, respectively. Comparison of transcription factor mRNA profiles from these cell populations with those previously seen in normal human bone marrow, demonstrated that although all of the 14 transcription factors described in vivo, could be detected during in vitro differentiation, vast differences in their expression profiles was observed. These data illustrate the limitations of cell lines as models for normal granulopoiesis.