Objective: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro-inflammatory and pro-apoptotic mediators, such as tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and Fas-Ligand (Fas-L). In this study, we evaluated the frequency of TNF-α, TGF-β1 and Fas-L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. Methods: The TNF-α−308G/A, TGF-β1 −509C/T, +869T/C, +915G/C, and Fas-L −844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well-defined area with genetically homogeneous population, using a polymerase chain reaction-based restriction fragment length polymorphism assay. Results: The mutant genotype C/T or T/T of TGF-β1 −509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild-type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18–27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF-β1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF-α−308G/A, TGF-β1 +869T/C and +915G/C and Fas-L -844T/C polymorphisms. Conclusions: The TGF-β1 −509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF-β1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.