<p>Esophageal squamous cell carcinoma (eSCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors such as nivolumab have shown clinical benefit, particularly in patients with high PD-L1 expression, this subgroup represents only a small fraction of eSCC cases. CDK4/6 inhibitors such as palbociclib have only been tested as second-line agents in eSCC, often in combination with EGFR inhibitors, with minimal benefit. Our study evaluates palbociclib as a first-line therapy in treatment-naive eSCC models. Using a panel of 22 eSCC cell lines with integrated multi-omics and phenotypic assays, we identified three response subtypes, resistant, delayed, and arrested, correlated to Rb-pathway status. Interestingly, in delayed responders, palbociclib treatment was associated with DNA damage accumulation and unprotected micronuclei enriched for cGAS, triggering activation of interferon-stimulated genes. Consistent with this, palbociclib enhanced immune cell infiltration in delayed eSCC spheroids within a preclinical vascularized 3D microfluidic system. Our study demonstrates that first-line palbociclib treatment unmasks intrinsic vulnerabilities in the CDK4/6-Rb axis and triggers innate immune activation in molecularly defined eSCC. Using a translationally relevant 3D vascularized microfluidic system, we provide evidence that early CDK4/6 inhibition not only stall cancer cell growth but also promotes immune cells recruitment. In conclusion, our study identifies palbociclib as a viable first-line therapeutic candidate in selected eSCC patients and uncover its immunomodulatory potential.</p>