Natural killer (NK) cells play a fundamental role in protecting the host against cancerous and virally infected cells, however the mechanisms that control NK cell maturation and Ly49 receptor expression are incompletely understood. We have identified a novel role for sclerostin domain-containing-1 (Sostdc1) in the regulation of NK maturation and cytotoxicity. Sostdc1-knockout (KO) mice display a partial NK maturation block of transitional (CD27+CD11b-) NK cells and display decreased frequencies of inhibitory Ly49G2 and increased frequencies of activating Ly49H, Ly49D receptors. We hypothesized that Ly49 receptors in Sostdc1-KO mice would correlate with high NK cell killing. Inversely, we observed Sostdc1-KO NK cells to be hyporesponsive towards β2m-KO targets in vitro and in vivo. Consistent with Sostdc1’s known role in Wnt signaling, we observed increased Tcf7 and Lef1 levels in Sostdc1-KO NK cells. Reciprocal bone marrow transplants show that Sostdc1 can regulate NK cells distinctly by nonhematopoietic stromal cells and hematopoietic cells. These data suggest niche cell involvement in NK cell behavior. Furthermore, we show evidence for progressive NK cell Ly49 developmental pathways in C57BL/6 (WT) mice that are not entirely stochastic. We analyzed NK cell “clusters” defined by combinatorial expression of activating Ly49H, Ly49D, and inhibitory Ly49G2, Ly49I receptors. Using the product rule to evaluate Ly49 receptor interdependencies, we found NK cells to tightly regulate Ly49 receptors at the iNK cell stage. We discovered non-random cluster development of Ly49I receptor by transplantation of sorted clusters. Comparing MHC-I-KO and WT mice, we found that MHC-I is a partial regulator of Ly49D and Ly49G2, even though no known MHC-I ligand is present in WT mice. Altogether, these data support a novel role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity and a regulatory mechanism for a non-stochastic expression for Ly49 receptors. These data provide important information for potential NK cell guidance for future NK cell immunotherapies.