Global gene-expression analysis established the temporal expression patterns and programs underlying the development of functional activity of ex vivo expanded (EXE) human granulocytes, as well as differences compared to peripheral blood (PB) granulocytes. CD34⁺ progenitor cells were cultured for 3 weeks to induce rapid expansion and granulocytic differentiation with 40% CD15⁺ cells by day 3 and 90% by day 12. Phagocytic and respiratory-burst activity increased with the fraction of CD15⁺⁺CD11b⁺ cells (myelocytes to segmented), and peaked by day 17. However, only 25% of CD15⁺⁺CD11b⁺ cells were phagocytic and respiratory-burst activity was 1/3 that of PB granulocytes. EXE granulocytes from later days and PB granulocytes showed similar expression of Fcg receptors (1A, 2A, 2C, 3A) and complement receptors (1, 3, 4). Later down-regulation of CD36 (expressed by macrophages) suggests lineage plasticity early in granulocytic differentiation. Expression in mature EXE and PB granulocytes was similar for most Fcg-receptor-mediated phagocytosis signaling proteins, including high-level expression of Hck, Fgr, and the actin-related-protein 2/3 complex. Lower expression of Lyn, Cdc42, pleckstrin, and PKC_I by EXE granulocytes may explain decreased phagocytosis. PB and mature EXE granulocytes expressed similar levels of NADPH-oxidase-complex genes and receptors for fMLP-mediated respiratory burst. Lower burst activity by EXE granulocytes may result from lower expression of Raf1 and PKC. Elevated expression of TLR2, TLR1, and CD14 in mature EXE and PB granulocytes supports a role for the TLR2 and CD14 pathway in zymosan-mediated respiratory-burst activity. Lower activity in EXE granulocytes may be due to greater expression of IRAK3, which inhibits TLR-mediated signaling.