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, alpha-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.