Abstract Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific member of the Hem-family of cytoplasmic adaptor proteins. Orthologs of Hem-1 in flies and worms are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem-1 are not known. Using a chemical mutagenesis strategy in mice to identify novel immune function genes, we positionally-cloned a nonsense mutation in the Hem1 gene. Loss of Hem-1 results in defective F-actin polymerization and actin capping in T lymphocytes and neutrophils, due to loss of the Rac GTPase-controlled actin-regulatory "WAVE" complex. T cell development and activation are impaired. Hem1-/- neutrophils are deficient in migration and phagocytic capacity. Conventional "B2" B cell development is inhibited at the pro-B and transitional T1 cell stages, and "B1" B cell development is also impaired. ELISA and ImageStream flow cytometric analyses reveal that NF-¿B-dependent transcription of proinflammatory cytokines, proceed normally in Hem1-/- mice, while TH17 differentiation is enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development and function by controlling cytoskeletal reorganization.