The molecular controls governing commitment of hematopoietic stem cells to specific lineages have still not been fully elucidated. The importance of understanding such controls is underscored by the fact that a block in differentiation is a hallmark of acute leukemias. Acute myeloid leukemia (AML), involving the precursors of myeloid cells (granulocytes and monocytes/macrophages), accounts for over 90 percent of acute leukemias in adults. In this application, I propose to continue our study of the molecular basis of differentiation of myeloid cells, employing a detailed analysis of the transcription factor PU.1, which regulates nearly every myeloid gene, and is absolutely required for normal myeloid development. PU.1 is expressed in stem cells and upregulated early during myeloid and lymphoid commitment. The importance of understanding how PU.1 is regulated is underscored by studies indicating that altered expression of PU. 1 can induce changes in hematopoietic lineage development, and in some experimental cases misexpression leads to leukemia. Thus, the overall goal of this continuation proposal is to continue our studies of how PU.1 is regulated, and what its function is in adult hematopoiesis. These studies will further our understanding of (1) commitment of normal hematopoietic precursors to the myeloid lineage; and (2) the block in normal myeloid maturation from blasts to mature myeloid cells in AML. Therefore, the Specific Aims are: (1)To determine how PU.1 expression is regulated in hematopoietic cells, using transgenic and knockout studies; (2) To determine the role of PU.1 in adult hematopoiesis by studying a conditional knockout model; and (3) To determine whether different levels of PU. 1 expression play a role in directing hematopoietic lineage determination and development.