Generation of mature T lymphocytes requires an intact hematopoietic stem cell compartment and functional thymic epithelium. We used the zebrafish (Danio rerio) to isolate mutations that affect the earliest steps in T lymphopoiesis and thymic organogenesis. Here we describe the results of a genetic screen in which gynogenetic diploid offspring from heterozygous females were analyzed by whole‐mount in situ hybridization for the expression of rag‐1. To assess immediately if a global defect in hematopoiesis resulted in the mutant phenotype, α‐embryonic globin expression was simultaneously assayed for multilineage defects. In this report, we present the results obtained with this strategy and show representative mutant phenotypes affecting early steps in T‐cell development and/or thymic epithelial cell development. We discuss the advantage of this strategy and the general usefulness of the zebrafish as a model system for vertebrate lymphopoiesis and thymic organogenesis. Developmental Dynamics 237:2575–2584, 2008. © 2008 Wiley‐Liss, Inc.