Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap₄A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap₄A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap₄A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap₄A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap₄A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.