Background: There has been little progress in the treatment of newly diagnosed glioblastoma in decades and standards of care for recurrent glioblastoma remain poorly defined. The multi-cyclin dependent kinase inhibitor Zotiraciclib (TG02) depletes short-lived survival proteins such as c-MYC and MCL-1 which are overexpressed in glioblastoma. The mode of action is thought to be specific inhibition of cyclin-dependent kinase (CDK) 9. Methods: EORTC 1608 was a three parallel group (A,B,C) phase Ib, non-randomized, multicenter study in isocitrate dehydrogenase wildtype newly diagnosed glioblastoma or anaplastic astrocytoma. In groups A and B, the maximum tolerated dose (MTD) of Zotiraciclib in elderly patients, in combination with radiotherapy alone (group A) or temozolomide alone (group B), stratified by O⁶-methylguanine DNA methyltransferase promoter methylation status, were determined. In group C, we explored single agent activity of TG02 at first relapse with a primary endpoint of progression-free survival at 6 months (PFS-6). Tumor expression of CDK-9, c-MYC and MCL-1 was determined by immunohistochemistry. Tumor-related extracellular vesicles were quantified according to the minimal information for studies of extracellular vesicles 2018 (MISEV2018) guidelines. Results: Main toxicities of zotiraciclib were neutropenia, gastrointestinal disorders and hepatotoxicity. The MTD was 150 mg twice weekly in group A (weekly) or twice weekly in alternating weeks in group B. PFS-6 in group C was 6.7%. The tumor expression of c-MYC and MCL-1 was moderately cross-correlated. High protein levels of MCL-1 were associated with inferior survival. The levels of tumor-related extracellular vesicles in the blood correlated with tumor volumes determined by MRI and increased from baseline to recurrence. Conclusions: Zotiraciclib exhibits overlapping toxicity with alkylating agents which are the cornerstone of medical treatment for glioblastoma. Its single agent clinical activity in the recurrent setting was low. The role of c-MYC and MCL-1 as therapeutic targets and the diagnostic value of extracellular vesicles in peripheral blood in glioblastoma warrant further study.