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Theimmune system functions to eradicate pathogens via well-coordinated interactions between a large diversity of cellular lineages. The development and functionality of all these immune cells must require precise calibration to maintain host defense and homeostasis. Even though a huge amount of molecules were validated in mouse models to regulate immunity, there still remains poorly understood how each component of the immune system such as T cells sense signals and sequentially respond or tolerate. Here we present a pipeline integrating high throughput immune-phenotyping and forward genetic screening with ENU mutagenesis, to delineate functions of genes in development and function in immune system of mammals. The immune-phenotyping not only involves surface staining of molecules but may incorporate intracellular assay of signal molecules on the single cell basis. It is important to note that forward genetic screening provide unbiased search of genetic regulators the functions of which could be completely unexpected. We identified a molecule termed as Rltpr, a key downstream node of the CD28 co-stimulation, by random mutagenesis on a sensitized background that causes a lethal lymphoproliferative disorder. Surprisingly, mutation of the novel gene Rltpr was sufficient to rescue the disease. We also identified multiple mutant lines with developmental defects such as those in neutrophils, B cells, NK cells and T cells. Dissection of genes at functional genomics level, is highly facilitated by the recent progress in high throughput immune-phenotyping with flow cytometric analysis up to 30 parameters, meanwhile genetic clues help decipher how the sophisticated system of immunity is maintained.