Telomere-biology disorders (TBDs) are characterized by short telomeres and defective telomere repair, and clinical manifestations include bone marrow failure (BMF), myeloid neoplasms, fibrotic lung disease and liver cirrhosis. CD4+ immunodeficiency has been described in patients with TBD, but no comprehensive profiling of immune cells has been published to date. The aim of this study was to deep-phenotype lymphocytes, monocytes, natural killer (NK) and dendritic cells (DCs), and to measure serum cytokines, chemokines and growth factors from patients harboring telomere-related pathogenic variants and exhibiting clinical features of TBDs. 20 patients and 10 healthy individuals were recruited, and mass cytometry (CyTOF) analysis of peripheral blood mononuclear cells (PBMCs) was employed. A total of 32 serum cytokines, chemokines and growth factors were simultaneously measured by Luminex™. CyTOF showed that TBD patients had lower frequency of CD4+ lymphocytes compared to controls (33 ± 14% vs. 47 ± 16%; p = 0.031), whereas the opposite was observed for CD8+ cells (54 ± 14% vs. 41 ± 11% in controls, p = 0.034). Accordingly, CD4/CD8 ratio was lower in patients (0.73 ± 0.56 vs. 1.3 ± 0.69 in controls, p = 0.050). B cells count was also decreased (4.8 x 104 ± 5.0 x 104/ 106 cells vs. 9.0 x 104 ± 3.4 x 104/ 106 cells in controls; p = 0.0047). Recent thymic emigrants (CD3+CD31+CD45RA+) and absolute counts of naïve CD4+, CD8+ and B cells were reduced (p < 0.01), reflecting the thymic involution and marrow insufficiency normally seen in telomeropathies. Activated effector memory and TEMRA CD4+ cells expressing exhaustion markers (C−D4+CD45RA+/−CCR7−CD38+HLADR+Ki67+PD-1+TIGITlow) and proliferating switched memory B cells (CD3−CD19+IgD−CD27+Ki67+) were higher (p = 0.046 and p = 0.041, respectively) suggesting the occurrence of an inflammatory milieu, supported by the prevalence of CD14+CD16+CXCR3+CCR4+ monocytes (2.6 ± 1.8% vs. 0.81 ± 0.28% in controls; p = 0.0042). Regarding T helper cells, TH1, TH17 and TH17.1 were decreased in patients (p < 0.05), whereas the TH2/TH1 ratio was two times higher (1.6 ± 0.83 vs. 0.82 ± 0.39 in controls; p = 0.013). Plasmacytoid DCs (CD3−CD141+CD123+CD1c−), known inducers of T helper differentiation, were reduced in patients (0.97 ± 0.55 vs. 2.0 ± 1.0% in controls; p = 0.014). An effector double negative T cell population (CD3+CD4−CD8−Vd2−CD45RA+CCR7−Tbet+Granzyme B+) was significantly increased (6.1 ± 6.0% vs. 1.3 ± 1.1% in controls; p = 0.00017). Circulating mucosal-associated invariant T (MAIT) cells (CD3+CD161highV7.2+) were markedly reduced (0.14 ± 0.18% vs. 2.8 ± 3.0% in controls; p < 0.0001) and, finally, a subset of immature NK cells (CD3−CD19−CD56brightCD16−CD27+) were more frequent in patients as compared to controls (p = 0.026). In addition, patients with TBD also display dysregulated cytokine levels. Telomere length positively correlated with angiopoietin-1, IL-1 superfamily, IL-3, IL-4, IL-7, IL-23, IL-27, M-CSF, MCP-1, MIP-3a and PDGF-BB, indicating that the shortest the telomere, the lowest the levels of these analytes. Taken together, our data show that TBDs feature a distinct immunophenotype, manifested through the depletion of CD4+ T and B cells, skewing of helper T cells, accumulation of activated subpopulations expressing inflammatory markers and a dramatic decrease of circulating MAIT cells. Serum cytokines levels are also disrupted, potentially due to the cellular immunity being disturbed.