Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry–based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206⁺) subsets and other CD206⁻ leukocytes. The CD206⁻ cells include: (1) three monocyte (CD14⁺) subsets, (2) CD11c⁺ dendritic cells (CD14⁻, CD11c⁺, HLA-DR⁺), (3) plasmacytoid dendritic cells (CD14⁻, CD11c⁻, HLA-DR⁺, CD123⁺), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169⁺ and CD169⁻ macrophages. In lung tissue, CD169⁻ macrophages were a prominent cell type. Using confocal microscopy, CD169⁺ macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169⁻ macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.