Extensive cell proliferation during embryogenesis often compromises genome integrity, increasing the risk of developmental defects. However, the mechanisms that safeguard genome integrity during this process remain poorly understood. Using early limb development as a model, we identify that DNA damage response factors are up-regulated in proliferating mesenchymal stem cells. Conditional knockout of Rpa1, a representative DNA damage response factor, in early limb bud mesenchyme results in the near-total absence of forelimbs and severely underdeveloped hindlimbs. Mechanistically, Rpa1 deletion leads to extensive DNA damage and activates the cGAS-STING pathway, driving transcription of Zbp1. Rpa1 deletion also leads to accumulation of Z-DNA bound by ZBP1, triggering the full activation of ZBP1 and subsequent mesenchymal stem cell death through PANoptosis. Our study reveals RPA1 as a vital protector of genomic stability during limb development and underscores ZBP1-dependent PANoptosis as a key pathway for eliminating cells with excessive DNA damage during embryonic development.