Objectives: NKG2D is an activating receptor expressed on different cells like NK cells, T cells and macrophages. It belongs to the C-type lectin-like family of receptors and interacts with H60, Rae1 and Mult1 ligands. NKG2D is a potent activating receptor on NK cells which acts as a molecular sensor for stress exposed cells expressing NKG2D ligands such as virally infected or tumor transformed cells. The aim of this work is to inactivate mouse NKG2D receptor in order to study its role in the development, activation, and homeostasis of NK cells in vitro and in vivo. Therefore, we have developed NKG2D deficient mouse strain after introducing mutations in Klrk1 gene by gene targeting. Although NKG2D is expressed on NK cell precursors (NKPs), it is not known whether NKG2D has the role in NK cell development. Therefore, our major goal was to investigate possible consequences of NKG2D-deficiency on the development, homeostasis and effector functions of NK cells. Methods: I have generated NKG2D-deficient mice by targeting the Klrk1 locus in ES cells derived from C57BL/6 mouse strain (Bruce 4). In this work I used flow cytometry analysis as well as different functional assays to analyse developmental NK cell subpopulations. I also tested effector functions of NKG2D-deficient NK cells in MCMV infection model. Results: Targeting vector for Klrk1 mutation was constructed and sequenced. Bruce 4 (C57/BL6) ES cells were transfected and picked up after positive (G418) and negative (gancyclovir) selection and screened for homologous recombinants by Southern blotting. Two clones were transiently transfected with pIC-Cre expressing vector to expel neoR cassette. G418 sensitive colonies were microinjected in blastocystas. NKG2D deficient mice mutant was made. Here I provide evidence for dual role of NKG2D in the physiology of NK cells: one, rather regulatory, implicated in their development, homeostasis and survival, and other, important for exerting their effector functions. We observed the following consequences of NKG2D deficiency on the NK cell development and homeostasis: a) faster division of NK cells, b) perturbation in size of NK cell subpopulations and, c) their augmented sensitivity to apoptosis. As expected, NKG2D deficient NK cells were considerably less responsive to tumor cell targets expressing NKG2D ligands, thus confirming the important role of NKG2D for exertion of their effector functions. However, Klrk1 -/- mice showed an enhanced NK cell-mediated resistance to MCMV infection as a consequence of NK cell dysregulation. Conclusion: Our study reveals a novel, yet unknown function of NKG2D in NK cell physiology. We have shown here for the first time, using genetically engineered mouse model, that NKG2D plays an important regulatory role in NK cell development, controlling proliferation, homeostasis and survival of NK cells.