The ATM-dependent DNA damage checkpoint plays a pivotal role in cellular response to ionizing radiation. Although amplification of the DNA damage signal through multifactorial protein complex formation of DNA damage checkpoint factors is crucial for proper DNA damage response in two-dimensionally cultured cells, the dynamics of the DNA damage response in three-dimensional tissues or organs remained to be determined. Here we used a model of reconstituted human skin and investigated the spatiotemporal dynamics of focus formation of DNA damage checkpoint factors after X irradiation. We found that DNA damage-induced foci were differentially formed in different layers. All cells in basal layers and approximately 40% of cells in spinous layers displayed foci. In basal cells, the foci showed linear dose relationships, and the number of foci decreased with increasing time after irradiation. We found that the initial foci grew within a few hours after irradiation, and persistent signals developed large foci. Colocalization of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 foci was detected, and all of them showed simultaneous focus growth, indicating amplification of DNA damage signals. These results confirmed a dynamic DNA damage response in three-dimensional tissue, which provides a practical model for studying DNA damage response in vivo.